Polypeptide

ABSTRACT

We disclose a PS4 variant polypeptide derivable from a parent polypeptide, the parent polypeptide having non-maltogenic exoamylase activity, which PS4 variant polypeptide comprises one or more of the following substitutions: G69P, A141P, G223A, A268P, G313P, S399P and G400P, with reference to the position numbering of a  Pseudomonas saccharophilia  exoamylase sequence shown as SEQ ID NO: 1. Such PS4 variant polypeptides may be used as exo-amylases, particularly as non-maltogenic exoamylases. Combinations of such PS4 variant polypeptides together with Novamyl are disclosed.

This application is a divisional of U.S. application Ser. No.10/864,874, filed Jun. 10, 2004, which claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 60/479,505, filed Jun. 19,2003, and under 35 U.S.C. § 119(a) to United Kingdom Application No. GB0313754.4, filed Jun. 13, 2003, each of which is expressly incorporatedby reference herein in its entirety.

FIELD

This invention relates to polypeptides, and nucleic acids encodingthese, and their uses as non-maltogenic exoamylases in producing foodproducts. In particular, the polypeptides are derived from polypeptideshaving non-maltogenic exoamylase activity, in particular, glucan1,4-alpha-maltotetrahydrolase (EC 3.2.1.60) activity.

SUMMARY

According to a first aspect of the present invention, we provide a PS4variant polypeptide derivable from a parent polypeptide, the parentpolypeptide having non-maltogenic exoamylase activity, which PS4 variantpolypeptide comprises one or more of the following substitutions: G69P,A141P, G223A, A268P, G313P, S399P and G400P, with reference to theposition numbering of a Pseudomonas saccharophilia exoamylase sequenceshown as SEQ ID NO: 1.

Preferably, the parent polypeptide comprises a non-maltogenicexoamylase. Preferably, the parent polypeptide comprises a glucan1,4-alpha-maltotetrahydrolase (EC 3.2.1.60). Preferably, the parentpolypeptide is or is derivable from Pseudomonas species, preferablyPseudomonas saccharophilia or Pseudomonas stutzeri. Preferably, theparent polypeptide is a non-maltogenic exoamylase from Pseudomonassaccharophilia having a sequence shown as SEQ ID NO: 1. Alternatively,or in addition, the parent polypeptide is a non-maltogenic exoamylasefrom Pseudomonas stutzeri having SWISS-PROT accession number P13507.

In preferred embodiments, the PS4 variant polypeptide has a higherthermostability compared to the parent polypeptide when tested under thesame conditions. Preferably, the half life (t_(1/2)), preferably at 60degrees C, is increased by 15% or more, preferably 50% or more, mostpreferably 100% or more, relative to the parent polypeptide. Preferably,the PS4 variant polypeptide has a higher pH stability compared to theparent polypeptide when tested under the same conditions. Preferably,the PS4 variant polypeptide has 10% or more, preferably 20% or more,preferably 50% or more, pH stability.

In preferred embodiments, the PS4 variant polypeptide comprises one ormore proline substitutions selected from the group consisting of: G69P,A141P, A268P, G313P, S399P and G400P, together with an alaninesubstitution G223A.

Preferably, it comprises a sequence selected from the group consistingof: PSac-69P (SEQ ID NO: 2), PSac-A141P (SEQ ID NO: 3), PSac-G223A (SEQID NO: 4), PSac-A268P (SEQ ID NO: 5), PSac-G313P (SEQ ID NO: 6),PSac-S399P (SEQ ID NO: 7), PSac-G400P (SEQ ID NO: 8), PStu-69P (SEQ IDNO: 12), PStu-A141P (SEQ ID NO: 13), PStu-G223A (SEQ ID NO: 14),PStu-A268P (SEQ ID NO: 15), PStu-G313P (SEQ ID NO: 16), PStu-S399P (SEQID NO: 17) and PStu-G400P (SEQ ID NO: 18).

There is provided, according to a second aspect of the presentinvention, a nucleic acid comprising a sequence capable of encoding aPS4 variant polypeptide according to the first aspect of the invention.

We provide, according to a third aspect of the present invention, a PS4nucleic acid sequence derivable from a parent sequence encoding apolypeptide having non-maltogenic exoamylase activity and comprising acodon encoding an amino acid at the specified position selected from thegroup consisting of: 69P, 141P, 223A, 268P, 313P, 399P and 400P, withreference to the position numbering of a Pseudomonas saccharophiliaexoamylase sequence shown as SEQ ID NO: 1.

As a fourth aspect of the present invention, there is provided a nucleicacid sequence derivable from a parent sequence, the parent sequencecapable of encoding a non-maltogenic exoamylase, which nucleic acidsequence comprises a substitution at one or more residues such that thenucleic acid encodes one or more of the following mutations at thepositions specified: 69P, 141P, 223A, 268P, 313P, 399P and 400P, withreference to the position numbering of a Pseudomonas saccharophiliaexoamylase sequence shown as SEQ ID NO: 1.

Preferably, the PS4 nucleic acid sequence is derived from a parentsequence encoding a non-maltogenic exoamylase by substitution of one ormore nucleotide residues.

Preferably, it comprises a codon CCA, CCC, CCG or CCT, at any one ormore of positions 207-209, 423-425, 804-806, 939-941, 1197-1199,1200-1202, and/or a codon GCA, GCC, GCG or GCT at positions 669-671,with reference to the position numbering of a Pseudomonas saccharophiliaexoamylase nucleotide sequence shown as SEQ ID NO: 10. Preferably, theparent sequence encodes a non-maltogenic exoamylase having a feature asset out above, or in which a polypeptide encoded by the nucleic acid hasany of the features as set out above.

We provide, according to a fifth aspect of the present invention, aplasmid comprising a PS4 nucleic acid according to the second, third orfourth aspect of the invention.

The present invention, in a sixth aspect, provides an expression vectorcomprising a PS4 nucleic acid to the second, third or fourth aspect ofthe invention, or capable of expressing a PS4 variant polypeptideaccording to the first aspect of the invention.

In a seventh aspect of the present invention, there is provided a hostcell comprising, preferably transformed with, a plasmid according to thefifth aspect of the sinvention or an expression vector according to thesixth aspect of the invention.

According to an eighth aspect of the present invention, we provide acell capable of expressing a polypeptide according to the first aspectof the invention.

Preferably, the cell or host cell is a bacterial, fungal or yeast cell.

We provide, according to a ninth aspect of the invention, a method ofexpressing a PS4 variant polypeptide, the method comprising obtaining ahost cell according to the seventh aspect of the invention, or a cellaccording to the eighth aspect of the invention, and expressing thepolypeptide from the cell or host cell, and optionally purifying thepolypeptide.

There is provided, in accordance with a tenth aspect of the presentinvention, a method of altering the sequence of a polypeptide byintroducing an amino acid substitution selected from the groupconsisting of: G69P, A141P, G223A, A268P, G313P, S399P and G400P (withreference to the position numbering of a Pseudomonas saccharophiliaexoamylase sequence shown as SEQ ID NO: 1), into a parent polypeptidehaving non-maltogenic exoamylase activity.

As an eleventh aspect of the invention, we provide a method of alteringthe sequence of a non-maltogenic exoamylase by introducing a G69P,A141P, G223A, A268P, G313P, S399P or G400P substitution, with referenceto the position numbering of a Pseudomonas saccharophilia exoamylasesequence shown as SEQ ID NO: 1.

Preferably, the sequence of the non-maltogenic exoamylase is altered byaltering the sequence of a nucleic acid which encodes the non-maltogenicexoamylase.

In highly preferred embodiments, the non-maltogenic exoamylase has afeature as set out above, or in which a polypeptide encoded by thenucleic acid has a feature as set out above.

We provide, according to a twelfth aspect of the invention, a method ofproducing a PS4 polypeptide variant, the method comprising introducingan amino acid substitution into a parent polypeptide havingnon-maltogenic exoamylase activity, the amino acid substitution beingselected from the group consisting of: G69P, A141P, G223A, A268P, G313P,S399P and G400P, with reference to the position numbering of aPseudomonas saccharophilia exoamylase sequence shown as SEQ ID NO: 1.

Preferably, the sequence of a nucleic acid encoding the parentpolypeptide is altered to introduce the amino acid substitution.Preferably, the parent sequence encodes a non-maltogenic exoamylase,preferably having a feature as set out above, or in which a polypeptideencoded by the nucleic acid has a feature as set out above.

According to a thirteenth aspect of the present invention, we provide amethod of altering the sequence of a nucleic acid encoding anon-maltogenic exoamylase, the method comprising introducing into thesequence a codon which encodes an amino acid residue selected from thegroup consisting of: 69P, 141P, 223A, 268P, 313P, 399P or 400P, withreference to the position numbering of a Pseudomonas saccharophiliaexoamylase sequence shown as SEQ ID NO: 1.

There is provided, according to a fourteenth aspect of the presentinvention, a method of increasing the thermostability, or the pHstability, or both, of a polypeptide, the method comprising the steps asset out above.

Preferably, the polypeptide is isolated or purified, or both.

We provide, according to a fifteenth aspect of the present invention, apolypeptide obtainable by a method according to any of the ninth tofourteenth aspects of the invention.

According to a sixteenth aspect of the present invention, we provide apolypeptide obtained by a method according to any of the ninth tofourteenth aspects of the invention.

According to a seventeenth aspect of the present invention, we provideuse of a PS4 variant polypeptide according to the first, sixteenth orseventeenth aspect of the invention, as an amylase.

We provide, according to an eighteenth aspect of the present invention,a process for treating a starch comprising contacting the starch with apolypeptide according to any of the first, sixteenth and seventeenthaspects of the invention, and allowing the polypeptide to generate fromthe starch one or more linear products.

According to a nineteenth aspect of the present invention, we provideuse of a PS4 variant polypeptide according to any of the first,sixteenth and seventeenth aspects of the invention, a nucleic acidaccording to the second, third, fourth or fifth aspect of the invention,a cell or a host cell according to the seventh or eighth aspect of theinvention, in preparing a food product.

As an twentieth aspect of the invention, we provide a process ofpreparing a food product comprising admixing a polypeptide according toany of the first, sixteenth and seventeenth aspects of the inventionwith a food ingredient.

In preferred embodiments, the food product is a dough product. Examplesinclude in general any processed dough product, including fried, deepfried, roasted, baked, steamed and boiled doughs, such as steamed breadand rice cakes. In highly preferred embodiments, the food product is abakery product.

According to an twenty-first aspect of the present invention, we providea process for making a bakery product comprising: (a) providing a starchmedium; (b) adding to the starch medium a PS4 variant polypeptideaccording to any of the first, sixteenth and seventeenth aspects of theinvention; and (c) applying heat to the starch medium during or afterstep (b) to produce a bakery product.

In preferred embodiments, the bakery product is a bread.

According to a twenty-second aspect of the present invention, we providea food product or a bakery product obtained by a process according toany of the nineteenth to twenty-first aspects of the invention.

Preferably, the food product is a dough or an animal feed.

According to a twenty-third aspect of the present invention, we providean improver composition for a dough, in which the improver compositioncomprises a PS4 variant polypeptide according to any of the first,sixteenth and seventeenth aspects of the invention and at least onefurther dough ingredient or dough additive.

According to a twenty-fourth aspect of the present invention, we providecomposition comprising a flour and a PS4 variant polypeptide accordingto any of the first, sixteenth and seventeenth aspects of the invention.

According to a twenty-fifth aspect of the present invention, we provideuse of a PS4 variant polypeptide according to any of the first,sixteenth and seventeenth aspects of the invention, in a bread productto retard or reduce detrimental staling, preferably starchretrogradation, of the bread product.

According to a twenty-sixth aspect of the present invention, we providean enzyme variant derivable from a parent enzyme, which parent enzyme isa member of the PS4 exoamylase family, in which the enzyme variantcomprises one or more amino acid modifications at one or more of thefollowing positions (using Pseudomonas saccharophilia exoamylase X16732numbering) relative to the parent enzyme: G69P, A141P, G223A, A268P,G313P, S399P and G400P or equivalent position(s) in other homologousmembers of the PS4 exoamylase family.

According to a twenty-seventh aspect of the present invention, weprovide a polypeptide being a variant of an exoamylase, whichpolypeptide comprises a sequence of an exoamylase together with aproline substitution at or about an amino acid position corresponding toposition 69, 141, 268, 313, 399 or 400, or an alanine substitution atposition 223, or both, of a Pseudomonas saccharophilia exoamylasesequence shown as SEQ ID NO: 1.

According to a twenty-eighth aspect of the present invention, we providea polypeptide sequence comprising a sequence of a non-maltogenicexoamylase which has been mutated to include a proline substitution atan amino acid position 69, 141, 268, 313, 399 or 400 or an alaninesubstitution at position 223, or both.

According to a twenty-ninth aspect of the present invention, we providea nucleic acid sequence derivable from a parent sequence encoding apolypeptide having non-maltogenic exoamylase activity and comprising acodon capable of encoding an amino acid substitution selected from thegroup consisting of: G69P, A141P, G223A, A268P, G313P, S399P and G400P,with reference to the position numbering of a Pseudomonas saccharophiliaexoamylase sequence shown as SEQ ID NO: 1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. The E. coli Bacillus shuttle vectors pCSmta and pCSmta-SBD withthe mta gene under control of the P32 promoter and the cgt signalsequence for extracellular expression of PS4 in B. subtilis.

FIG. 2. Plasmids used in the Quick Exchange method. Mutations were madeusing one of the SDM vectors, and after confirmation of the desiredmutation by sequencing, the fragment was placed back into thecorresponding pCSA vector. Restriction sites used for exchanging thefragments are indicated.

FIG. 3. Antistaling effect of PS4 variants with mutations A141P andG223A in comparison with wild type PS4 (PS4ccl) measured by DifferentialScanning Calorimetry (DSC).

SEOUENCE LISTINGS

SEQ ID NO: 1 shows a PS4 reference sequence, derived from Pseudomonassaccharophila maltotetrahydrolase amino acid sequence.

SEQ ID NO: 2 shows the sequence of PSac-G69P; Pseudomonas saccharophilamaltotetrahydrolase amino acid sequence with P substitution for Gresidue at position 69. SEQ ID NO: 3 shows the sequence of PSac-A141P;Pseudomonas saccharophila maltotetrahydrolase amino acid sequence with Psubstitution for A residue at position 141. SEQ ID NO: 4 shows thesequence of PSac-G223A; Pseudomonas saccharophila maltotetrahydrolaseamino acid sequence with A substitution for G residue at position 223

SEQ ID NO: 5 shows the sequence of PSac-A268P; Pseudomonas saccharophilamaltotetrahydrolase amino acid sequence with P substitution for Aresidue at position 268. SEQ ID NO: 6 shows the sequence of PSac-G313P;Pseudomonas saccharophila maltotetrahydrolase amino acid sequence with Psubstitution for G residue at position 313. SEQ ID NO: 7 shows thesequence of PSac-S399P; Pseudomonas saccharophila maltotetrahydrolaseamino acid sequence with P substitution for S residue at position 399.SEQ ID NO: 8 shows the sequence of PSac-G400P; Pseudomonas saccharophilamaltotetrahydrolase amino acid sequence with P substitution for Gresidue at position 400.

SEQ ID NO: 9 shows an amino acid sequence of Pseudomonas saccharophilamaltotetrahydrolase. Pseudomonas saccharophila Glucan1,4-alpha-maltotetrahydrolase precursor (EC 3.2.1.60) (G4-amylase)(Maltotetraose-forming amylase) (Exo-maltotetraohydrolase)(Maltotetraose-forming exo-amylase). SWISS-PROT accession number P22963.

SEQ ID NO: 10 shows a nucleic acid sequence of Pseudomonas saccharophilamaltotetrahydrolase. P. saccharophila mta gene encodingmaltotetraohydrolase (EC number =3.2.1.60). GenBank accession numberX16732.

SEQ ID NO: 11 shows an amino acid sequence of Pseudomonas stutzerimaltotetrahydrolase.

SEQ ID NO: 12 shows the sequence of PStu-69P; Pseudomonas stutzerimaltotetrahydrolase amino acid sequence with P substitution for Gresidue at position 69. SEQ ID NO: 13 shows the sequence of PStu-A141P;Pseudomonas stutzeri maltotetrahydrolase amino acid sequence with Psubstitution for A residue at position 141. SEQ ID NO: 14 shows thesequence of PStu-G223A; Pseudomonas stutzeri maltotetrahydrolase aminoacid sequence with A substitution for G residue at position 223.

SEQ ID NO: 15 shows the sequence of PStu-A268P; Pseudomonas stutzerimaltotetrahydrolase amino acid sequence with P substitution for Aresidue at position 268. SEQ ID NO: 16 shows the sequence of PStu-G313P;Pseudomonas stutzeri maltotetrahydrolase amino acid sequence with Psubstitution for G residue at position 313. SEQ ID NO: 17 shows thesequence of PStu-S399P; Pseudomonas stutzeri maltotetrahydrolase aminoacid sequence with P substitution for S residue at position 399. SEQ IDNO: 18 shows the sequence of PStu-G400P; Pseudomonas stutzerimaltotetrahydrolase amino acid sequence with P substitution for Gresidue at position 400.

SEQ ID NO: 19 shows the sequence of Pseudomonas stutzeri (Pseudomonasperfectomarina). Glucan 1,4-alpha-maltotetrahydrolase precursor (EC3.2.1.60) (G4-amylase) (Maltotetraose-forming amylase)(Exo-maltotetraohydrolase)(Maltotetraose-forming exo-amylase).SWISS-PROT accession number P113507.

SEQ ID NO: 20 shows the sequence of Pseudomonas stutzerimaltotetrahydrolase nucleic acid sequence. P. stutzerimaltotetraose-forming amylase (amyP) gene, complete cds. GenBankaccession number M24516.

DETAILED DESCRIPTION

PS4 Variants

We provide for polypeptides, and nucleic acids encoding these, which arevariants of polypeptides having non-maltogenic exoamylase activity. Suchvariant polypeptides are referred to in this document as “PS4 variantpolypeptides”, and the nucleic acids as “PS4 variant nucleic acids”. PS4variant polypeptides and nucleic acids will be described in furtherdetail below.

Specifically, we provide for PS4 variant polypeptides with sequencealterations comprising proline or alanine substitutions, or both, in anon-maltogenic exoamylase sequence.

Such variant polypeptides retain at least some of the features of theparent polypeptides, and additionally preferably have additionalbeneficial properties, for example, enhanced activity orthermostability, or pH resistance, or any combination (preferably all).The PS4 substitution mutants described here may preferably be used forany purpose for which the parent enzyme is suitable. In particular, theymay be used in any application for which exo-maltotetraohydrolase isused. In highly preferred embodiments, they have the added advantage ofhigher thermostability, or higher pH stability, or both. Examples ofsuitable uses for the PS4 variant polypeptides and nucleic acids includefood production, in particular baking, as well as production offoodstuffs and feedstuffs; further examples are set out in detail below.

The “parent” sequences, i.e., the sequences on which the PS4 variantpolypeptides and nucleic acids are based, preferably are polypeptideshaving non-maltogenic exoamylase activity. The terms “parent enzymes”and “parent polypeptides” should be interpreted accordingly, and takento mean the enzymes and polypeptides on which the PS4 variantpolypeptides are based.

In particularly preferred embodiments, the parent sequences arenon-maltogenic exoamylase enzymes, preferably bacterial non-maltogenicexoamylase enzymes. In highly preferred embodiments, the parent sequencecomprises a glucan 1,4-alpha-maltotetrahydrolase (EC 3.2.1.60).Preferably, the parent sequence is from Pseudomonas species, for examplePseudomonas saccharophilia or Pseudomonas stutzeri.

In preferred embodiments, the parent polypeptide comprises, or ishomologous to, a Pseudomonas saccharophilia non-maltogenic exoamylasehaving a sequence shown as SEQ ID NO: 1. Proteins and nucleic acidsrelated to, preferably having sequence or functional homology withPseudomonas saccharophilia non-maltogenic exoamylase Pseudomonassaccharophilia exoamylase sequence shown as SEQ ID NO: 1 are referred toin this document as members of the “PS4 family”. Examples of “PS4family” non-maltogenic exoamylase enzymes suitable for use in generatingthe PS4 variant polypeptides and nucleic acids are disclosed in furtherdetail below.

In some preferred embodiments, the parent polypeptide comprises anon-maltogenic exoamylase from Pseudomonas saccharophilia non-maltogenicexoamylase having a sequence shown as SEQ ID NO: 1, or a SWISS-PROTaccession number P22963. In other preferred embodiments, the parentpolypeptide comprises a non-maltogenic exoamylase from Pseudomonasstutzeri having a sequence shown as SEQ ID NO: 11, or a Pseudomonasstutzeri non-maltogenic exoamylase having SWISS-PROT accession numberP13507.

The PS4 variant polypeptides and nucleic acids vary from their parentsequences by including one or more mutations. In other words, thesequence of the PS4 variant polypeptide or nucleic acid is differentfrom that of its parent at one or more positions or residues. Inpreferred embodiments, the mutations comprise amino acid substitutions,that is, a change of one amino acid residue for another. Thus, the PS4variant polypeptides comprise one or more changes in the nature of theamino acid residue at one or more positions of the parent sequence.

In describing the different PS4 variant polypeptide variants produced orwhich are contemplated to be encompassed by this document, the followingnomenclature will be adopted for ease of reference: [original aminoacid/position according to the numbering system/substituted amino acid].Accordingly, for example, the substitution of alanine with proline inposition 141 is designated as A141P. Multiple mutations are separated byslash marks “/”, e.g. A141P/G223A representing mutations in position 141and 223 substituting alanine with proline and glycine with alaninerespectively.

All positions referred to in the present document by numbering refer tothe numbering of a Pseudomonas saccharophilia exoamylase referencesequence shown below (SEQ ID NO: 1):   1 DQAGKSPAGV RYHGGDEIILQGFHWNVVRE APNDWYNILR QQASTIAADG FSAIWMPVPW  61 RDFSSWTD

G KSGGGEGYFW HDFNKNGRYG SDAQLRQAAG ALGGAGVKVL YDVVPNHMNR 121 GYPDKEINLPAGQGFWRNDC

DPGNYPNDC DDGDRFIGGE SDLNTGHPQI YGMFRDELAN 181 LRSGYGAGGF RFDFVRGYAPERVDSWMSDS ADSSFCVGEL WK

PSEYPSW DWRNTASWQQ 241 IIKDWSDRAK CPVFDFALKE RMQNGSV

DW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG 301 QNGGQHHWAL QD

LIRQAYA YILTSPGTPV VYWSHMYDWG YGDFIRQLIQ VRRTAGVRAD 361 SAISFHSGYSGLVATVSGSQ QTLVVALNSD LANPGQVA

 SFSEAVNASN GQVRVWRSGS 421 GDGGGNDGGE GGLVNVNFRC DNGVTQMGDS VYAVGNVSQLGNWSPASAVR LTDTSSYPTW 481 KGSIALPDGQ NVEWKCLIRN EADATLVRQW QSGGNNQVQAAAGASTSGSF

The reference sequence is derived from the Pseudomonas saccharophiliasequence having SWISS-PROT accession number P22963, but without thesignal sequence MSHILRAAVLAAVLLPFPALA (SEQ ID NO: 21).

The PS4 variant polypeptide variants described here preferably compriseone or more amino acid substitutions selected from the group consistingof 69P, A141P, G223A, A268P, G313P, S399P and G400P. In one embodiment,the PS4 variants are derived from a Pseudomonas saccharophilanon-maltogenic enzyme sequence. Accordingly, and preferably, the PS4variant polypeptide variants are preferably selected from the groupconsisting of: PSac-69P (SEQ ID NO: 2), PSac-A141P (SEQ ID NO: 3),PSac-G223A (SEQ ID NO: 4), PSac-A268P (SEQ ID NO: 5), PSac-G313P (SEQ IDNO: 6), PSac-S399P (SEQ ID NO: 7) and PSac-G400P (SEQ ID NO: 8).

In highly preferred embodiments, we provide for a PSac-A141P PS4 variantpolypeptide having a sequence shown as SEQ ID NO: 3.

In another embodiment, the PS4 variants are derived from a Pseudomonasstutzeri non-maltogenic enzyme sequence, preferably shown as SEQ ID NO:11 below:   1 DQAGKSPNAV RYHGGDEIIL QGFHWNVVRE APNDWYNILR QQAATIAADGFSAIWMPVPW  61 RDFSSWSDGS KSGGGEGYFW HDFNKNGRYG SDAQLRQAAS ALGGAGVKVLYDVVPNHMNR 121 GYPDKEINLP AGQGFWRNDC ADPGNYPNDC DDGDRFIGGD ADLNTGHPQVYGMFRDEFTN 181 LRSQYGAGGF RFDFVRGYAP ERVNSWMTDS ADNSFCVGEL WKGPSEYPNWDWRNTASWQQ 241 IIKDWSDRAK CPVFDFALKE RMQNGSIADW KHGLNGNPDP RWREVAVTFVDNHDTGYSPG 301 QNGGQHHWAL QDGLIRQAYA YILTSPGTPV VYWSHMYDWG YGDFIRQLIQVRRAAGVRAD 361 SAISFHSGYS GLVATVSGSQ QTLVVALNSD LGNPGQVASG SFSEAVNASNGQVRVWRSGT 421 GSGGGEPGAL VSVSFRCDNG ATQMGDSVYA VGNVSQLGNW SPAAALRLTDTSGYPTWKGS 481 IALPAGQNEE WKCLIRNEAN ATQVRQWQGG ANNSLTPSEG ATTVGRL

Accordingly, and preferably, the PS4 polypeptide variants are preferablyselected from the group consisting of: PStu-69P (SEQ ID NO: 12),PStu-A141P (SEQ ID NO: 13), PStu-G223A (SEQ ID NO: 14), PStu-A268P (SEQID NO: 15), PStu-G313P (SEQ ID NO: 16), PStu-S399P (SEQ ID NO: 17) andPStu-G400P (SEQ ID NO: 18).

In highly preferred embodiments, we provide for a PStu-A141P PS4 variantpolypeptide having a sequence shown as SEQ ID NO: 13.

In the context of the present description a specific numbering of aminoacid residue positions in PS4 exoamylase enzymes is employed.Preferably, all positions referred to in the present document bynumbering refer to the numbering of a Pseudomonas saccharophiliaexoamylase reference sequence shown below (SEQ ID NO: 1).

In this respect, by alignment of the amino acid sequences of variousknown exoamylases it is possible to unambiguously allot a exoamylaseamino acid position number to any amino acid residue position in anyexoamylase enzyme, the amino acid sequence of which is known.

Using this numbering system originating from for example the amino acidsequence of the exoamylase obtained from Pseudomonas saccharophilia,aligned with amino acid sequences of a number of other known exoamylase,it is possible to indicate the position of an amino acid residue in aexoamylase unambiguously.

Therefore, the numbering system, even though it may use a specificsequence as a base reference point, is also applicable to all relevanthomologous sequences. For example, the position numbering may be appliedto homologous sequences from other Pseudomonas species, or homologoussequences from other bacteria. Preferably, such homologous have 60% orgreater homology, for example 70% or more, 80% or more, 90% or more or95% or more homology, with the reference sequence SEQ ID NO: I above, orthe sequences having SWISS-PROT accession numbers P22963 or P13507,preferably with all these sequences. Sequence homology between proteinsmay be ascertained using well known alignment programs and hybridisationtechniques described herein. Such homologous sequences will be referredto in this document as the “PS4 Family”.

Furthermore, and as noted above, the numbering system used in thisdocument makes reference to a reference sequence SEQ ID NO: 1, which isderived from the Pseudomonas saccharophilia sequence having SWISS-PROTaccession number P22963, but without the signal sequenceMSHILRAAVLAAVLLPFPALA (SEQ ID NO: 21). This signal sequence is located Nterminal of the reference sequence and consists of 21 amino acidresidues. Accordingly, it will be trivial to identify the particularresidues to be mutated or substituted in corresponding sequencescomprising the signal sequence, or indeed, corresponding sequencescomprising any other N- or C- terminal extensions or deletions. Forexample, the sequence of Pseudomonas saccharophilia non-maltogenicexoamylase having SWISS-PROT accession number P22963 or a Pseudomonasstutzeri non-maltogenic exoamylase having SWISS-PROT accession numberP13507.

The PS4 variant polypeptides may comprise one or more of the mutationsset out above; in some aspects, there is just one mutation. In otheraspects there are two mutations. In particular, they may comprise anypair of such mutations. In further aspects there are three mutations. Inother aspects there are more than three mutations. They may comprise anycombination of three, four, five, six, seven or more of the mutations asset out.

In particularly preferred embodiments, where there is more than onemutation, the PS4 variant polypeptides comprise the substitutions A141Por G223A, or both. We therefore disclose PS4 variant polypeptides whichcomprise A141P together with at least one other mutation. We alsodisclose PS4 variant polypeptides which comprise G223A together with atleast one other mutation. In particular, we disclose a PS4 variantpolypeptide comprising A141P as well as G223A, i.e., A141P/G223P.

Other mutations, such as deletions, insertions, substitutions,transversions, transitions and inversions, at one or more otherlocations, may also be included.

We also provide PS4 nucleic acids having sequences which correspond toor encode the alterations in the PS4 variant polypeptide sequences. Theskilled person will be aware of the relationship between nucleic acidsequence and polypeptide sequence, in particular, the genetic code andthe degeneracy of this code, and will be able to construct such PS4nucleic acids without difficulty. For example, he will be aware that foreach amino acid substitution in the PS4 variant polypeptide sequence,there may be one or more codons which encode the substitute amino acid.Accordingly, it will be evident that, depending on the degeneracy of thegenetic code with respect to that particular amino acid residue, one ormore PS4 nucleic acid sequences may be generated corresponding to thatPS4 variant polypeptide sequence. Furthermore, where the PS4 variantpolypeptide comprises more than one substitution, for exampleA141P/G223A, the corresponding PS4 nucleic acids may comprise pairwisecombinations of the codons which encode respectively the two amino acidchanges.

Thus, for example, PS4 variant nucleic acid sequences may comprise acodon CCA, CCC, CCG or CCT, at any one or more of positions 207-209,423-425, 804-806, 939-941, 1197-1199, 1200-1202, and/or a codon GCA,GCC, GCG or GCT at positions 669-671, with reference to the positionnumbering of a Pseudomonas saccharophilia exoamylase nucleotide sequencewith GenBank accession number X16732, or as shown as SEQ ID NO: 10.Preferably, the position numbering is with reference to the sequenceshown as SEQ ID NO: 10. Corresponding Pseudomonas stutzerinon-maltogenic exoamylase variants can similarly be constructed.

It will be understood that nucleic acid sequences which are notidentical to the particular PS4 variant nucleic acid sequences, but arerelated to these, will also be useful for the methods and compositionsdescribed here. Accordingly, it will be understood that the followingare included: (a) a nucleotide sequence that is a variant, homologue,derivative or fragment of a PS4 variant nucleic acid sequence; (b) anucleotide sequence that is the complement of a PS4 variant nucleic acidsequence; (c) a nucleotide sequence that is the complement of a variant,homologue, derivative or fragment of a PS4 variant nucleic acidsequence; (d) a nucleotide sequence that is capable of hybridising to aPS4 variant nucleic acid sequence; (e) a nucleotide sequence that iscapable of hybridising to a variant, homologue, derivative or fragmentof a PS4 variant nucleic acid sequence; (f) a nucleotide sequence thatis the complement of a nucleotide sequence that is capable ofhybridising to a PS4 variant nucleic acid sequence; (g) a nucleotidesequence that is the complement of a nucleotide sequence that is capableof hybridising to a variant, homologue, derivative or fragment of a PS4variant nucleic acid sequence; (h) a nucleotide sequence that is capableof hybridising to the complement of a PS4 variant nucleic acid sequence;and (i) a nucleotide sequence that is capable of hybridising to thecomplement of a variant, homologue, derivative or fragment of a PS4variant nucleic acid sequence. Unless the context dictates otherwise,the term “PS4 variant nucleic acid” should be taken to include each ofthese entities listed above.

Mutations in amino acid sequence and nucleic acid sequence may be madeby any of a number of techniques, as known in the art. In particularlypreferred embodiments, the mutations are introduced into parentsequences by means of PCR (polymerase chain reaction) using appropriateprimers, as illustrated in the Examples. It is therefore possible toalter the sequence of a polypeptide by introducing an amino acidsubstitution selected from the group consisting of: G69P, A141P, G223A,A268P, G313P, S399P and G400P into a parent polypeptide havingnon-maltogenic exoamylase activity, into a Pseudomonas saccharophilia ora Pseudomonas stutzeri exoamylase sequence at amino acid or nucleic acidlevel, as described. However, as noted elsewhere, this sequence does notitself need to comprise a wild type sequence; rather, it can be analready mutated sequence.

Furthermore, it will of course be appreciated that the PS4 variantpolypeptide does not need in fact to be actually derived from a wildtype polypeptide or nucleic acid sequence by, for example, step by stepmutation. Rather, once the sequence of the PS4 variant polypeptide isestablished, the skilled person can easily make that sequence from thewild type with all the mutations, via means known in the art, forexample, using appropriate oligonucleotide primers and PCR. In fact, thePS4 variant polypeptide can be made de novo with all its mutations,through, for example, peptide synthesis methodology.

In general, however, the PS4 variant polypeptides and/or nucleic acidsare derived or derivable from a “precursor” sequence. The term“precursor” as used herein means an enzyme that precedes the enzymewhich is modified according to the methods and compositions describedhere. Thus, the precursor may be an enzyme that is modified bymutagenesis as described elsewhere in this document. Likewise, theprecursor may be a wild type enzyme, a variant wild type enzyme or analready mutated enzyme.

We further describe a method in which the sequence of a non-maltogenicexoamylase is altered by altering the sequence of a nucleic acid whichencodes the non-maltogenic exoamylase.

The PS4 variant polypeptides and nucleic acids may be produced by anymeans known in the art. Specifically, they may be expressed fromexpression systems, which may be in vitro or in vivo in nature.Specifically, we provide for plasmids and expression vectors comprisingPS4 nucleic acid sequences, preferably capable of expressing PS4 variantpolypeptides. Cells and host cells which comprise and are preferablytransformed with such PS4 nucleic acids, plasmids and vectors are alsodisclosed, and it should be made clear that these are also encompassedin this document.

In preferred embodiments, the PS4 variant nucleic acid or polypeptidesequence is in an isolated form. The term “isolated” means that thesequence is at least substantially free from at least one othercomponent with which the sequence is naturally associated in nature andas found in nature. In one aspect, preferably the sequence is in apurified form. The term “purified” means that the sequence is in arelatively pure state—e.g. at least about 90% pure, or at least about95% pure or at least about 98% pure.

We further disclose a construct comprising a PS4 variant nucleotidesequence; a vector comprising PS4 variant nucleotide sequence; a plasmidcomprising PS4 variant nucleotide sequence; a transformed cellcomprising a PS4 variant nucleotide sequence; a transformed tissuecomprising a PS4 variant nucleotide sequence; a transformed organcomprising a PS4 variant nucleotide sequence; a transformed hostcomprising a PS4 variant nucleotide sequence; a transformed organismcomprising a PS4 variant nucleotide sequence. We also disclose methodsof expressing a PS4 variant nucleotide sequence, such as expression in ahost cell; including methods for transferring same. We also disclosemethods of isolating the PS4 variant nucleotide sequence, such asisolating from a host cell.

Other aspects concerning a variant PS4 amino acid sequence include: aconstruct encoding a variant PS4 amino acid sequence; a vector encodinga variant PS4 amino acid sequence; a plasmid encoding a variant PS4amino acid sequence; a transformed cell expressing a variant PS4 aminoacid sequence; a transformed tissue expressing a variant PS4 amino acidsequence; a transformed organ expressing a variant PS4 amino acidsequence; a transformed host expressing a variant PS4 amino acidsequence; a transformed organism expressing a variant PS4 amino acidsequence. We also disclose methods of purifying the amino acid sequencefor use in the methods and compositions described here, such asexpression in a host cell; including methods of transferring same, andthen purifying said sequence.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA and immunology, which are within thecapabilities of a person of ordinary skill in the art. Such techniquesare explained in the literature. See, for example, J. Sambrook, E. F.Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual,Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel,F. M. et al. (1995 and periodic supplements; Current Protocols inMolecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York,N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation andSequencing: Essential Techniques, John Wiley & Sons; J. M. Polak andJames O'D. McGee, 1990, In Situ Hybridization: Principles and Practice;Oxford University Press; M. J. Gait (Editor), 1984, OligonucleotideSynthesis: A Practical Approach, Irl Press; D. M. J. Lilley and J. E.Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A:Synthesisand Physical Analysis of DNA Methods in Enzymology, Academic Press;Using Antibodies: A Laboratory Manual: Portable Protocol NO. I by EdwardHarlow, David Lane, Ed Harlow (1999, Cold Spring Harbor LaboratoryPress, ISBN 0-87969-544-7); Antibodies: A Laboratory Manual by Ed Harlow(Editor), David Lane (Editor) (1988, Cold Spring Harbor LaboratoryPress, ISBN 0-87969-314-2), 1855, Lars-Inge Larsson“Immunocytochemistry: Theory and Practice”, CRC Press inc., Baca Raton,Fla., 1988, ISBN 0-8493-6078-1, John D. Pound (ed); “ImmunochemicalProtocols, vol 80”, in the series: “Methods in Molecular Biology”,Humana Press, Totowa, N.J., 1998, ISBN 0-89603-493-3, Handbook of DrugScreening, edited by Ramakrishna Seethala, Prabhavathi B. Fernandes(2001, New York, N.Y., Marcel Dekker, ISBN 0-8247-0562-9); and Lab Ref:A Handbook of Recipes, Reagents, and Other Reference Tools for Use atthe Bench, Edited Jane Roskams and Linda Rodgers, 2002, Cold SpringHarbor Laboratory, ISBN 0-87969-630-3. Each of these general texts isherein incorporated by reference.

Parent Enzyme

The PS4 variant polypeptides are derived from, or are variants of,another sequence, known as a “parent enzyme”, a “parent polypeptide” ora “parent sequence”.

The term “parent enzyme” as used in this document means the enzyme thathas a close, preferably the closest, chemical structure to the resultantvariant, i.e., the PS4 variant polypeptide or nucleic acid. The parentenzyme may be a precursor enzyme (i.e. the enzyme that is actuallymutated) or it may be prepared de novo. The parent enzyme may be a wildtype enzyme.

The term “precursor” as used herein means an enzyme that precedes theenzyme which is modified to produce the enzyme. Thus, the precursor maybe an enzyme that is modified by mutagenesis. Likewise, the precursormay be a wild type enzyme, a variant wild type enzyme or an alreadymutated enzyme.

The term “wild type” is a term of the art understood by skilled personsand means a phenotype that is characteristic of most of the members of aspecies occurring naturally and contrasting with the phenotype of amutant. Thus, in the present context, the wild type enzyme is a form ofthe enzyme naturally found in most members of the relevant species.Generally, the relevant wild type enzyme in relation to the variantpolypeptides described here is the most closely related correspondingwild type enzyme in terms of sequence homology.

However, where a particular wild type sequence has been used as thebasis for producing a variant PS4 polypeptide as described here, thiswill be the corresponding wild type sequence regardless of the existenceof another wild type sequence that is more closely related in terms ofamino acid sequence homology.

The parent enzyme is preferably a polypeptide which preferably exhibitsnon-maltogenic exoamylase activity. Preferably, the parent enzyme is anon-maltogenic exoamylase itself. For example, the parent enzyme may bea Pseudomonas saccharophila non-maltogenic exoamylase, such as apolypeptide having SWISS-PROT accession number P22963, or a Pseudomonasstutzeri non-maltogenic exoamylase, such as a polypeptide havingSWISS-PROT accession number P13507. Other members of the PS4 family maybe used as parent enzymes; such PS4 family members will generally besimilar to, homologous to, or functionally equivalent to either of thesetwo enzymes, and may be identified by standard methods, such ashybridisation screening of a suitable library using probes, or by genomesequence analysis.

In particular, functional equivalents of either of these two enzymes, aswell as other members of the “PS4 family” may also be used as startingpoints or parent polypeptides for the generation of PS4 variantpolypeptides as described here.

The term “functional equivalent” in relation to a parent enzyme being aPseudomonas saccharophila non-maltogenic exoamylase, such as apolypeptide having SWISS-PROT accession number P22963, or a Pseudomonasstutzeri non-maltogenic exoamylase, such as a polypeptide havingSWISS-PROT accession number P13507 means that the functional equivalentcould be obtained from other sources. The functionally equivalent enzymemay have a different amino acid sequence but will have in general havesome non-maltogenic exoamylase activity.

In highly preferred embodiments, the functional equivalent will havesequence homology to either of the Pseudomonas saccharophila andPseudomonas stutzeri non-maltogenic exoamylases mentioned above,preferably both. The functional equivalent may also have sequencehomology with any of the sequences set out as SEQ ID NOs: 1 to 20,preferably SEQ ID NO: 1 or SEQ ID NO: 11 or both. Sequence homologybetween such sequences is preferably at least 60%, preferably 65% ormore, preferably 75% or more, preferably 80% or more, preferably 85% ormore, preferably 90% or more, preferably 95% or more. Such sequencehomologies may be generated by any of a number of computer programsknown in the art, for example BLAST or FASTA, etc. A suitable computerprogram for carrying out such an alignment is the GCG Wisconsin Bestfitpackage (University of Wisconsin, U.S.A; Devereux et al., 1984, NucleicAcids Research 12:387). Examples of other software than can performsequence comparisons include, but are not limited to, the BLAST package(see Ausubel et al., 1999 ibid - Chapter 18), FASTA (Atschul et al.,1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparisontools. Both BLAST and FASTA are available for offline and onlinesearching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). Howeverit is preferred to use the GCG Bestfit program.

In other embodiments, the functional equivalents will be capable ofspecifically hybridising to any of the sequences set out above. Methodsof determining whether one sequence is capable of hybridising to anotherare known in the art, and are for example described in Sambrook, et al(supra) and Ausubel, F. M. et al. (supra). In highly preferredembodiments, the functional equivalents will be capable of hybridisingunder stringent conditions, e.g. 65° C. and 0.1×SSC {1×SSC=0.15 M NaCl,0.015 M Na₃ Citrate pH 7.0}.

The parent enzymes may be modified at the amino acid level or thenucleic acid level to generate the PS4 variant sequences described here.Therefore, we provide for the generation of G69P, A141P, G223A, A268P,G313P, S399P or G400P amino acid sequence variants (i.e., PS4 variantpolypeptides), by introducing one or more corresponding codon changes inthe nucleotide sequence encoding a non-maltogenic exoamylasepolypeptide. Preferably, such changes include one or more of codonsubstitutions CCA, CCC, CCG or CCT, at any one or more of positions207-209, 423-425, 804-806, 939-941, 1197-1199, 1200-1202, and/or a codonGCA, GCC, GCG or GCT at positions 669-671, in any PS4 family nucleicacid sequence, for example, a Pseudomonas saccharophila or a Pseudomonasstutzeri non-maltogenic exoamylase nucleic acid sequence (e.g., X16732or M24516).

The nucleic acid numbering should preferably be with reference to theposition numbering of a Pseudomonas saccharophilia exoamylase nucleotidesequence shown as SEQ ID NO: 10. Alternatively, or in addition,reference may be made to the sequence with GenBank accession numberX16732. In preferred embodiments, the nucleic acid numbering should bewith reference to the nucleotide sequence shown as SEQ ID NO: 10.However, as with amino acid residue numbering, the residue numbering ofthis sequence is to be used only for reference purposes only. Inparticular, it will be appreciated that the above codon changes can bemade in any PS4 family nucleic acid sequence. For example, sequencechanges can be made to a Pseudomonas saccharophila or a Pseudomonasstutzeri non-maltogenic exoamylase nucleic acid sequence (e.g., X16732,SEQ ID NO: 10 or M24516, SEQ ID NO: 20).

Amylase

The PS4 variant polypeptides generally comprise amylase activity.

The term “amylase” is used in its normal sense—e.g. an enzyme that isinter alia capable of catalysing the degradation of starch. Inparticular they are hydrolases which are capable of cleaving α-D-(1→4)O-glycosidic linkages in starch.

Amylases are starch-degrading enzymes, classified as hydrolases, whichcleave α-D-(1→4) O-glycosidic linkages in starch. Generally, α-amylases(E.C. 3.2.1.1, α-D-(1→4)-glucan glucanohydrolase) are defined asendo-acting enzymes cleaving α-D-(1→4) O-glycosidic linkages within thestarch molecule in a random fashion. In contrast, the exo-actingamylolytic enzymes, such as β-amylases (E.C. 3.2.1.2, α-D-(1→4)-glucanmaltohydrolase), and some product-specific amylases cleave the starchmolecule from the non-reducing end of the substrate. β-Amylases,α-glucosidases (E.C. 3.2.1.20, α-D-glucoside glucohydrolase),glucoamylase (E.C. 3.2.1.3, α-D-(1→4)-glucan glucohydrolase), andproduct-specific amylases can produce malto-oligosaccharides of aspecific length from starch.

Non-maltogenic Exoamylase

The PS4 variant polypeptides described in this document are derived from(or variants of) polypeptides which preferably exhibit non-maltogenicexoamylase activity. Preferably, these parent enzymes are non-maltogenicexoamylases themselves. The PS4 variant polypeptides themselves inhighly preferred embodiments also exhibit non-maltogenic exoamylaseactivity.

In highly preferred embodiments, the term “non-maltogenic exoamylaseenzyme” as used in this document should be taken to mean that the enzymedoes not initially degrade starch to substantial amounts of maltose asanalysed in accordance with the product determination procedure asdescribed in this document.

In highly preferred embodiments, the non-maltogenic exoamylase comprisesan exo-maltotetraohydrolase. Exo-maltotetraohydrolase (E.C.3.2.1.60) ismore formally known as glucan 1,4-alpha-maltotetrahydrolase. This enzymehydrolyses 1,4-alpha-D-glucosidic linkages in amylaceous polysaccharidesso as to remove successive maltotetraose residues from the non-reducingchain ends.

Assays for Non-maltogenic Exoamylase Activity

The following system is used to characterize polypeptides havingnon-maltogenic exoamylase activity which are suitable for use accordingto the methods and compositions described here. This system may forexample be used to characterise the PS4 parent or variant polypeptidesdescribed here.

By way of initial background information, waxy maize amylopectin(obtainable as WAXILYS 200 from Roquette, France) is a starch with avery high amylopectin content (above 90%). 20 mg/ml of waxy maize starchis boiled for 3 min. in a buffer of 50 mM MES(2-(N-morpholino)ethanesulfonic acid), 2 mM calcium chloride, pH 6.0 andsubsequently incubated at 50∇C and used within half an hour.

One unit of the non-maltogenic exoamylase is defined as the amount ofenzyme which releases hydrolysis products equivalent to 1 μmol ofreducing sugar per min. when incubated at 50 degrees C. in a test tubewith 4 ml of 10 mg/ml waxy maize starch in 50 mM MES, 2 mM calciumchloride, pH 6.0 prepared as described above. Reducing sugars aremeasured using maltose as standard and using the dinitrosalicylic acidmethod of Bernfeld, Methods Enzymol., (1954), 1, 149-158 or anothermethod known in the art for quantifying reducing sugars.

The hydrolysis product pattern of the non-maltogenic exoamylase isdetermined by incubating 0.7 units of non-maltogenic exoamylase for 15or 300 min. at 50∇C in a test tube with 4 ml of 10 mg/ml waxy maizestarch in the buffer prepared as described above. The reaction isstopped by immersing the test tube for 3 min. in a boiling water bath.

The hydrolysis products are analyzed and quantified by anion exchangeHPLC using a Dionex PA 100 column with sodium acetate, sodium hydroxideand water as eluents, with pulsed amperometric detection and with knownlinear maltooligosaccharides of from glucose to maltoheptaose asstandards. The response factor used for maltooctaose to maltodecaose isthe response factor found for maltoheptaose.

Preferably, the PS4 parent polypeptides (and the PS4 variantpolypeptides) have non-maltogenic exoamylase activity such that if anamount of 0.7 units of said non-maltogenic exoamylase were to incubatedfor 15 minutes at a temperature of 50° C. at pH 6.0 in 4 ml of anaqueous solution of 10 mg preboiled waxy maize starch per ml bufferedsolution containing 50 mM 2-(N-morpholino)ethane sulfonic acid and 2 mMcalcium chloride then the enzyme would yield hydrolysis product(s) thatwould consist of one or more linear malto-oligosaccharides of from twoto ten D-glucopyranosyl units and optionally glucose; such that at least60%, preferably at least 70%, more preferably at least 80% and mostpreferably at least 85% by weight of the said hydrolysis products wouldconsist of linear maltooligosaccharides of from three to tenD-glucopyranosyl units, preferably of linear maltooligosaccharidesconsisting of from four to eight D-glucopyranosyl units.

For ease of reference, and for the present purposes, the feature ofincubating an amount of 0.7 units of the non-maltogenic exoamylase for15 minutes at a temperature of 50° C. at pH 6.0 in 4 ml of an aqueoussolution of 10 mg preboiled waxy maize starch per ml buffered solutioncontaining 50 mM 2-(N-morpholino)ethane sulfonic acid and 2 mM calciumchloride, may be referred to as the “Waxy Maize Starch Incubation Test”.

The hydrolysis products can be analysed by any suitable means. Forexample, the hydrolysis products may be analysed by anion exchange HPLCusing a Dionex PA 100 column with pulsed amperometric detection andwith, for example, known linear maltooligosaccharides of from glucose tomaltoheptaose as standards.

As used herein, the term “linear malto-oligosaccharide” is used in thenormal sense as meaning 2-10 units of α-D-glucopyranose linked by anα-(1→4) bond.

Thermostability and pH Stability

Preferably, the PS4 variant polypeptide is thermostable; preferably, ithas higher thermostability than its parent enzyme.

As used herein the term ‘thermostable’ relates to the ability of theenzyme to retain activity after exposure to elevated temperatures.Preferably, the PS4 variant polypeptide is capable of degradingresistant starch at temperatures of from about 20° C. to about 50° C.Suitably, the enzyme retains its activity after exposure to temperaturesof up to about 95° C.

The thermostability of an enzyme such as a non-maltogenic exoamylase ismeasured by its half life. Thus, the PS4 variant polypeptides describedhere have half lives extended relative to the parent enzyme bypreferably 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% ormore. A PS4 variant polypeptide may be one in particular in which thehalf life (t_(1/2)) is increased by 15% or more, preferably 50% or more,most preferably 100% or more, relative to the parent polypeptide,preferably when tested under the same conditions.

As used here, the half life (t½) is the time (in minutes) during whichhalf the enzyme activity is inactivated under defined heat conditions.In preferred embodiments, the half life is assayed at 60 degrees C.Preferably, the sample is heated for 1-10 minutes at 60° C. or higher.The half life value is then calculated by measuring the residual amylaseactivity, by any of the methods described here. Preferably, a half lifeassay is conducted as described in more detail in the Examples.

Preferably, the PS4 variant polypeptide is pH stable; more preferably,it has a higher pH stability than its cognate parent polypeptide. Asused herein the term ‘pH stable’ relates to the ability of the enzyme toretain activity over a wide range of pHs. Preferably, the PS4 variantpolypeptide is capable of degrading resistant starch at a pH of fromabout 5 to about 10.5. In one embodiment, the degree of pH stability maybe assayed by measuring the half life of the enzyme in specific pHconditions. In another embodiment, the degree of pH stability may beassayed by measuring the activity or specific activity of the enzyme inspecific pH conditions. The specific pH conditions may be any pH frompH5 to pH10.5.

Thus, the PS4 variant polypeptide may have a longer half life, or ahigher activity (depending on the assay) when compared to the parentpolypeptide under identical conditions. The PS4 variant polypeptides mayhave 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or longerhalf life when compared to their parent polypeptides under identical pHconditions. Alternatively, or in addition, they may have such higheractivity when compared to the parent polypeptide under identical pHconditions.

Uses of PS4 Variant Polypeptides and Nucleic Acids

ThePS4 variant polypeptides, nucleic acids, host cells, expressionvectors, etc, may be used in any application for which an amylase may beused. In particular, they may be used to substitute for anynon-maltogenic exoamylase. They may be used to supplement amylase ornon-maltogenic exoamylase activity, whether alone or in combination withother known amylases or non-maltogenic exoamylases.

The PS4 variant sequences described here may be used in variousapplications in the food industry—such as in bakery and drink products,they may also be used in other applications such as a pharmaceuticalcomposition, or even in the chemical industry. In particular, the PS4variant polypeptides and nucleic acids are useful for various industrialapplications including baking (as disclosed in WO 99/50399), flourstandardisation (volume enhancement or improvement) as well as feedprocessing (see below). They may be used to produce maltotetraose fromstarch and other substrates.

The PS4 variant polypeptides may be used to enhance the volume of foods,including baked foods such as bread. Thus, food products comprising ortreated with PS4 variant polypeptides are expanded in volume whencompared to products which have not been so treated, or treated withparent polypeptides. In other words, the food products have a largervolume of air per volume of food product. Alternatively, or in addition,the food products treated with PS4 variant polypeptides have a lowerdensity, or weight (or mass) per volume ratio. In particularly preferredembodiments, the PS4 variant polypeptides are used to enhance the volumeof bread. Volume enhancement or expansion is beneficial because itreduces the gumminess or starchiness of foods. Light foods are preferredby consumers, and the customer experience is enhanced. In preferredembodiments, the use of PS4 variant polypeptides enhances the volume by10%, 20%, 30% 40%, 50% or more.

The use of PS4 variant polypeptides to increase the volume of foods isdescribed in detail in Example 10.

Food Uses

The PS4 variant polypeptides and nucleic acids described here may beused as—or in the preparation of—a food. Here, the term “food” is usedin a broad sense—and covers food for humans as well as food for animals(i.e. a feed). In a preferred aspect, the food is for human consumption.The food may be in the from of a solution or as a solid—depending on theuse and/or the mode of application and/or the mode of administration.

The PS4 variant polypeptides and nucleic acids may be used as a foodingredient. As used herein the term “food ingredient” includes aformulation, which is or can be added to functional foods or foodstuffsand includes formulations which can be used at low levels in a widevariety of products that require, for example, acidifying oremulsifying. The food ingredient may be in the from of a solution or asa solid—depending on the use and/or the mode of application and/or themode of administration.

The PS4 variant polypeptides and nucleic acids disclosed here may be -or may be added to—food supplements. The PS4 variant polypeptides andnucleic acids disclosed here may be—or may be added to—functional foods.As used herein, the term “functional food” means food which is capableof providing not only a nutritional effect and/or a taste satisfaction,but is also capable of delivering a further beneficial effect toconsumer. Although there is no legal definition of a functional food,most of the parties with an interest in this area agree that they arefoods marketed as having specific health effects.

The PS4 variant polypeptides may also be used in the manufacture of afood product or a foodstuff. Typical foodstuffs, which also includefeedstuffs such as animal feed, include dairy products, meat products,poultry products, fish products and bakery products. Preferably, thefoodstuff is a bakery product. Typical bakery (baked) products includebread—such as loaves, rolls, buns, pizza bases etc.—Danish pastry,pretzels, tortillas, cakes, cookies, biscuits, krackers etc.

Retrogradation/Staling

The PS4 variant polypeptides may in general be used in foodapplications, for example, in the production of foodstuffs, inparticular starch products such as baked starch products. In onespecific embodiment, the PS4 variant polypeptides are used to preventdetrimental degradation of starch media, in particular, starch gels. Inone preferred aspect, we describe the use of such PS4 variant proteinsthat are capable of retarding the staling of starch. The PS4 variantpolypeptides are also capable of retarding the detrimentalretrogradation of starch media, such as starch gels.

Most starch granules are composed of a mixture of two polymers: anessentially linear polysaccharide called amylose and a highly branchedpolysaccharide called amylopectin. Amylopectin is a very large, branchedmolecule consisting of chains of α-D-glucopyranosyl units joined by (14) linkages, wherein said chains are attached by α-D-(1→6) linkages toform branches. Amylopectin is present in all natural starches,constituting about 75% of most common starches. Starches consistingentirely of amylopectin are known as waxy starches, e.g. waxy corn (waxymaize). Amylose is essentially a linear chain of (1Π4) linkedα-D-glucopyranosyl units having few α-D-(1Π6) branches. Most starchescontain about 25% amylose.

Starch granules heated in the presence of water undergo anorder-disorder phase transition called gelatinization, where liquid istaken up by the swelling granules. Gelatinization temperatures vary fordifferent starches and depend for the native, unmodified starches ontheir biological source. Upon cooling of freshly baked bread the amylosefraction, within hours, retrogrades to develop a network. This processis beneficial in that it creates a desirable crumb structure with a lowdegree of firmness and improved slicing properties. More graduallycrystallisation of amylopectin takes place within the gelatinised starchgranules during the days after baking. In this process amylopectin isbelieved to reinforce the amylose network in which the starch granulesare embedded. This reinforcement leads to increased firmness of thebread crumb. This reinforcement is one of the main causes of breadstaling.

As a consequence of detrimental retrogradation, the water-holdingcapacity of the paste or gel system is changed with importantimplications on the gel texture and dietary properties. It is known thatthe quality of baked bread products gradually deteriorates duringstorage. The crumb loses softness and elasticity and becomes firm andcrumbly. This so-called staling is primarily due to the detrimentalretrogradation of starch, which is understood to be a transition of thestarch gelatinised during baking from an amorphous state to a quasicrystalline state. The increase in crumb firmness is often used as ameasure of the staling process of bread.

The rate of detrimental retrogradation or crystallisation of amylopectindepends on the length of the side chains of amylopectin. In accordancewith this, cereal amylopectin retrogrades at a slower rate thanamylopectin from pea or potato, which has a longer average chain lengththan cereal amylopectin. Thus, enzymatic hydrolysis of the amylopectinside chains, for example, by PS4 variant polypeptides havingnon-maltogenic exoamylase activity, can markedly reduce theircrystallisation tendencies.

Accordingly, the use of PS4 variant polypeptides as described here whenadded to the starch at any stage of its processing into a food product,e.g., before during or after baking into bread can retard or impede orslow down the retrogradation. Such use is described in further detailbelow.

Assays for Measurement of Retrogradation (Inc. Staling)

For evaluation of the antistaling effect of the PS4 variant polypeptideshaving non-maltogenic exoamylase activity described here, the crumbfirmness can be measured 1, 3 and 7 days after baking by means of anInstron 4301 Universal Food Texture Analyzer or similar equipment knownin the art.

Another method used traditionally in the art and which is used toevaluate the effect on starch retrogradation of a PS4 variantpolypeptide having non-maltogenic exoamylase activity is based on DSC(differential scanning calorimetry). Hereby the melting enthalpy ofretrograded amylopectin in bread crumb or crumb from a model systemdough baked with or without enzymes (control) is measured. The DSCequipment applied in the described examples is a Mettler-Toledo DSC 820run with a temperature gradient of 10° C. per min. from 20 to 95° C. Forpreparation of the samples 10-20 mg of crumb are weighed and transferredinto Mettler-Toledo aluminium pans which then are hermetically sealed.

The model system doughs used in the described examples contain standardwheat flour and optimal amounts of water or buffer with or without thenon-maltogenic PS4 variant exoamylase. They are mixed in a 10 or 50 gBrabender Farinograph for 6 or 7 min., respectively. Samples of thedoughs are placed in glass test tubes (15*0.8 cm) with a lid. These testtubes are subjected to a baking process in a water bath starting with 30min. incubation at 33° C. followed by heating from 33 to 95° C. with agradient of 1.1° C. per min. and finally a 5 min. incubation at 95° C.Subsequently, the tubes are stored in a thermostat at 20° C. prior toDSC analysis.

Preparation of Starch Products

We provide the use of PS4 variant polypeptides in the preparation offood products, in particular, starch products. The method comprisesforming the starch product by adding a non-maltogenic exoamylase enzymesuch as a PS4 variant polypeptide, to a starch medium. If the starchmedium is a dough, then the dough is prepared by mixing together flour,water, the non-maltogenic exoamylase which is a PS4 variant polypeptideand optionally other possible ingredients and additives.

The term “starch” should be taken to mean starch per se or a componentthereof, especially amylopectin. The term “starch medium” means anysuitable medium comprising starch. The term “starch product” means anyproduct that contains or is based on or is derived from starch.Preferably, the starch product contains or is based on or is derivedfrom starch obtained from wheat flour. The term “flour” as used hereinis a synonym for the finely-ground meal of wheat or other grain.Preferably, however, the term means flour obtained from wheat per se andnot from another grain. Thus, and unless otherwise expressed, referencesto “wheat flour” as used herein preferably mean references to wheatflour per se as well as to wheat flour when present in a medium, such asa dough.

A preferred flour is wheat flour or rye flour or mixtures of wheat andrye flour. However, dough comprising flour derived from other types ofcereals such as for example from rice, maize, barley, and durra are alsocontemplated. Preferably, the starch product is a bakery product. Morepreferably, the starch product is a bread product. Even more preferably,the starch product is a baked farinaceous bread product. The term “bakedfarinaceous bread product” is understood to refer to any baked productbased on ground cereals and baked on a dough obtainable by mixing flour,water, and a leavening agent under dough forming conditions. Furthercomponents can of course be added to the dough mixture.

Thus, if the starch product is a baked farinaceous bread product (whichis a highly preferred embodiment), then the process comprises mixing—inany suitable order—flour, water, and a leavening agent under doughforming conditions and further adding a PS4 variant polypeptide,optionally in the form of a premix. The leavening agent may be achemical leavening agent such as sodium bicarbonate or any strain ofSaccharomyces cerevisiae (Baker's Yeast).

The PS4 variant non-maltogenic exoamylase can be added together with anydough ingredient including the water or dough ingredient mixture or withany additive or additive mixture. The dough can be prepared by anyconventional dough preparation method common in the baking industry orin any other industry making flour dough based products.

Baking of farinaceous bread products such as for example white bread,bread made from bolted rye flour and wheat flour, rolls and the like istypically accomplished by baking the bread dough at oven temperatures inthe range of from 180 to 250° C. for about 15 to 60 minutes. During thebaking process a steep temperature gradient (200→120° C.) is prevailingin the outer dough layers where the characteristic crust of the bakedproduct is developed. However, owing to heat consumption due to steamgeneration, the temperature in the crumb is only close to 100° C. at theend of the baking process.

We therefore describe a process for making a bread product comprising:(a) providing a starch medium; (b) adding to the starch medium a PS4variant polypeptide as described in this document; and (c) applying heatto the starch medium during or after step (b) to produce a breadproduct. We also describe a process for making a bread productcomprising adding to a starch medium a PS4 variant polypeptide asdescribed.

The non-maltogenic exoamylase PS4 variant polypeptide can be added as aliquid preparation or as a dry pulverulent composition either comprisingthe enzyme as the sole active component or in admixture with one or moreadditional dough ingredient or dough additive.

In order to improve further the properties of the baked product andimpart distinctive qualities to the baked product further doughingredients and/or dough additives may be incorporated into the dough.Typically, such further added components may include dough ingredientssuch as salt, grains, fats and oils, sugar, dietary fibre substances,milk powder, gluten and dough additives such as emulsifiers, otherenzymes, hydrocolloids, flavouring agents, oxidising agents, mineralsand vitamins.

The emulsifiers are useful as dough strengtheners and crumb softeners.As dough strengtheners, the emulsifiers can provide tolerance withregard to resting time and tolerance to shock during the proofing.Furthermore, dough strengtheners will improve the tolerance of a givendough to variations in the fermentation time. Most dough strengthenersalso improve on the oven spring which means the increase in volume fromthe proofed to the baked goods. Lastly, dough strengtheners willemulsify any fats present in the recipe mixture.

The crumb softening, which is mainly a characteristic of themonoglycerides, is attributed to an interaction between the emulsifierand the amylose fraction of the starch leading to formation of insolubleinclusion complexes with the amylose which will not recrystallize uponcooling and which will not therefore contribute to firmness of the breadcrumb.

Improving Composition

We describe improver compositions, which include bread improvingcompositions and dough improving compositions. These comprise a PS4variant polypeptide, optionally together with other ingredients such asan emulsifying agent, or a further enzyme, or both. The further enzymesmay comprise one or more of: an oxidase, a lipase and a xylanase.

We also provide for the use of such a bread and dough improvingcompositions in baking. In a further aspect, we provide a baked productor dough obtained from the bread improving composition or doughimproving composition. In another aspect, we describe a baked product ordough obtained from the use of a bread improving composition or a doughimproving composition.

Dough Preparation

A dough may be prepared by admixing flour, water, a dough improvingcomposition comprising PS4 variant polypeptide (as described above) andoptionally other ingredients and additives.

The dough improving composition can be added together with any doughingredient including the flour, water or optional other ingredients oradditives. The dough improving composition can be added before the flouror water or optional other ingredients and additives. The doughimproving composition can be added after the flour or water, or optionalother ingredients and additives. The dough can be prepared by anyconventional dough preparation method common in the baking industry orin any other industry making flour dough based products.

The dough improving composition can be added as a liquid preparation orin the form of a dry powder composition either comprising thecomposition as the sole active component or in admixture with one ormore other dough ingredients or additive.

The amount of the PS4 variant polypeptide non-maltogenic exoamylase thatis added is normally in an amount which results in the presence in thefinished dough of 50 to 100,000 units per kg of flour, preferably 100 to50,000 units per kg of flour. Preferably, the amount is in the range of200 to 20,000 units per kg of flour.

In the present context, 1 unit of the non-maltogenic exoamylase isdefined as the amount of enzyme which releases hydrolysis productsequivalent to 1 μmol of reducing sugar per min. when incubated at 50∇Cin a test tube with 4 ml of 10 mg/ml waxy maize starch in 50 mM MES, 2mM calcium chloride, pH 6.0 as described hereinafter.

The dough as described here generally comprises wheat meal or wheatflour and/or other types of meal, flour or starch such as corn flour,corn starch, maize flour, rice flour, rye meal, rye flour, oat flour,oat meal, soy flour, sorghum meal, sorghum flour, potato meal, potatoflour or potato starch. The dough may be fresh, frozen, or part-baked.

The dough may be a leavened dough or a dough to be subjected toleavening. The dough may be leavened in various ways, such as by addingchemical leavening agents, e.g., sodium bicarbonate or by adding aleaven (fermenting dough), but it is preferred to leaven the dough byadding a suitable yeast culture, such as a culture of Saccharomycescerevisiae (baker's yeast), e.g. a commercially available strain of S.cerevisiae.

The dough may also comprise other conventional dough ingredients, e.g.:proteins, such as milk powder, gluten, and soy; eggs (either whole eggs,egg yolks or egg white); an oxidant such as ascorbic acid, potassiumbromate, potassium iodate, azodicarbonamide (ADA) or ammoniumpersulfate; an amino acid such as L-cysteine; a sugar; a salt such assodium chloride, calcium acetate, sodium sulfate or calcium sulfate.

The dough may comprise fat such as granulated fat or shortening. Thedough may further comprise a further emulsifier such as mono- ordiglycerides, sugar esters of fatty acids, polyglycerol esters of fattyacids, lactic acid esters of monoglycerides, acetic acid esters ofmonoglycerides, polyoxethylene stearates, or lysolecithin.

We also describe a pre-mix comprising flour together with thecombination as described herein. The pre-mix may contain otherdough-improving and/or bread-improving additives, e.g. any of theadditives, including enzymes, mentioned herein. For some applications,the flour dough preferably comprises a hard flour.

The term “hard flour” as used herein refers to flour which has a higherprotein content such as gluten than other flours and is suitable for theproduction of, for example, bread. The term “hard flour” as used hereinis synonymous with the term “strong flour”.

A preferred flour for some applications is wheat flour. However doughscomprising flour derived from, for example, maize, corn, oat, barley,rye, durra, rice, soy, sorghum and potato are also contemplated.Preferably the flour dough comprises a hard wheat flour.

Further Dough Additives and Ingredients

One or more additives or further ingredients may be added to the dough.

Typically, further dough additives or ingredients (components) includeconventionally used dough additives or ingredients such as salt,sweetening agents such as sugars, syrups or artificial sweeteningagents, lipid substances including shortening, margarine, butter or ananimal or vegetable oil, glycerol and one or more dough additives suchas emulsifying agents, starch degrading enzymes, cellulose orhemicellulose degrading enzymes, proteases, non-specific oxidisingagents such as those mentioned above, flavouring agents, lactic acidbacterial cultures, vitamins, minerals, hydrocolloids such as alginates,carrageenans, pectins, vegetable gums including e.g. guar gum and locustbean gum, and dietary fibre substances.

Suitable emulsifiers which may be used as further dough additivesinclude lecithin, polyoxyethylene stearat, mono- and diglycerides ofedible fatty acids, acetic acid esters of mono- and diglycerides ofedible fatty acids, lactic acid esters of mono- and diglycerides ofedible fatty acids, citric acid esters of mono- and diglycerides ofedible fatty acids, diacetyl tartaric acid esters of mono- anddiglycerides of edible fatty acids, sucrose esters of edible fattyacids, sodium stearoyl-2-lactylate, and calcium stearoyl-2-lactylate.

The further dough additive or ingredient can be added together with anydough ingredient including the flour, water or optional otheringredients or additives, or the dough improving composition. Thefurther dough additive or ingredient can be added before the flour,water, optional other ingredients and additives or the dough improvingcomposition. The further dough additive or ingredient can be added afterthe flour, water, optional other ingredients and additives or the doughimproving composition.

The further dough additive or ingredient may conveniently be a liquidpreparation. However, the further dough additive or ingredient may beconveniently in the form of a dry composition.

Preferably the further dough additive or ingredient is selected from thegroup consisting of a vegetable oil, a vegetable fat, an animal fat,shortening, glycerol, margarine, butter, butterfat and milk fat.

Preferably the further dough additive or ingredient is at least 1% theweight of the flour component of dough. More preferably, the furtherdough additive or ingredient is at least 2%, preferably at least 3%,preferably at least 4%, preferably at least 5%, preferably at least 6%.If the additive is a fat, then typically the fat may be present in anamount of from 1 to 5%, typically 1 to 3%, more typically about 2%.

Further Enzyme

In addition to the PS4 variant polypeptides, one or more further enzymesmay be used, for example added to the dough preparation, foodstuff orstarch composition or feed.

Other enzymes which are useful as further dough additives include asexamples oxidoreductases, maltose oxidising enzymes, glucose oxidase,hexose oxidase, pyranose oxidase and ascorbate oxidase, hydrolases, suchas lipases (see below) and esterases as well as glycosidases likeα-amylase, pullulanase, and xylanase. Oxidoreductases, such as forexample glucose oxidase and hexose oxidase, can be used for doughstrengthening and control of volume of the baked products and xylanasesand other hemicellulases may be added to improve dough handlingproperties, crumb softness and bread volume. Lipases are useful as doughstrengtheners and crumb softeners and α-amylases and other amylolyticenzymes may be incorporated into the dough to control bread volume andfurther reduce crumb firmness. Further details of lipases are set outbelow.

Further enzymes that may be used may be selected from the groupconsisting of a xylanase, a cellulase, a hemicellulase, a starchdegrading enzyme, a protease, a lipoxygenase, an oxidoreductase, such asa maltose oxidising enzyme, a lipase and an oxidising enzyme such as anyone or more of glucose oxidase (EC 1.1.3.4), carbohydrate oxidase,glycerol oxidase, pyranose oxidase (EC 1.1.3.10) and hexose oxidase (EC1.1.3.5).

Among starch degrading enzymes, amylases are particularly useful asdough improving additives. α-amylase breaks downs starch into dextrinswhich are further broken down by β-amylase to maltose. Other usefulstarch degrading enzymes which may be added to a dough compositioninclude glucoamylases and pullulanases.

Preferably, the further enzyme is at least a xylanase and/or at least anamylase. The term “xylanase” as used herein refers to xylanases (EC3.2.1.32) which hydrolyse xylosidic linkages.

The term “amylase” as used herein refers to amylases such as α-amylases(EC 3.2.1.1), which hydrolyse 1,4-α-D-glucosidic linkages inpolysaccharides containing three or more 1,4-α-linked glucose units,β-amylases (EC 3.2.1.2) which hydrolyse 1,4-α-D-glucosidic linkages inpolysaccharides so as to remove successive maltose units from thenon-reducing ends of the chains, and γ-amylases (EC 3.2.1.3) whichhydrolyse the terminal 1,4-linked α-D-glucose residues successively fromnon-reducing ends of chains with the release of α-D-glucose.

The further enzyme can be added together with any dough ingredientincluding the flour, water or optional other ingredients or additives,or the dough improving composition. The further enzyme can be addedbefore the flour, water, and optionally other ingredients and additivesor the dough improving composition. The further enzyme can be addedafter the flour, water, and optionally other ingredients and additivesor the dough improving composition. The further enzyme may convenientlybe a liquid preparation. However, the composition may be conveniently inthe form of a dry composition.

Some enzymes of the dough improving composition are capable ofinteracting with each other under the dough conditions to an extentwhere the effect on improvement of the rheological and/or machineabilityproperties of a flour dough and/or the quality of the product made fromdough by the enzymes is not only additive, but the effect issynergistic.

In relation to improvement of the product made from dough (finishedproduct), it may be found that the combination results in a substantialsynergistic effect in respect to crumb homogeneity as defined herein.Also, with respect to the specific volume of baked product a synergisticeffect may be found.

The further enzyme may include a “lipase”. The term “lipase” as usedherein refers to enzymes which are capable of hydrolysing carboxylicester bonds to release carboxylate (EC 3.1.1). Examples of lipasesinclude but are not limited to triacylglycerol lipase (EC 3.1.1.3),galactolipase (EC 3.1.1.26), phospholipase Al (EC 3.1.1.32) andphospholipase A2 (EC 3.1.1.4).

The lipase may be isolated and/or purified from natural sources or itmay be prepared by use of recombinant DNA techniques. Preferably thelipase is selected from the group comprising triacylglycerol lipase, agalactolipase, phospholipase. In another aspect, the lipase(s) may beone or more of the following: triacylglycerol lipase (EC 3.1.1.3),phospholipase A2 (EC 3.1.1.4), galactolipase (EC 3.1.1.26),phospholipase A1 (EC 3.1.1.32), lipoprotein lipase A2 (EC 3.1.1.34).Lipases are also known as lipolytic enzymes.

Suitable lipases for use in as further enzymes include (but are notlimited to) one or more lipase selected from the lipases disclosed inEP0130064, WO 98/26057, WO00/32758,, WO 02/03805, and LipopanH, alsoreferred to as Lecitase UltraTM and HL1232 (LipopanH is disclosed inGRAS Notice 000103, copies of which are available from the Department ofHealth & Human Services, Food & Drug Administration, Washington DC20204). Each of these references is incorporated herein by reference.

The lipase may in some applications suitably be LipopanF (supplied byNovozymes) or a variant, derivative or homologue thereof.

Other Uses

The PS4 variants are suitable for the production of maltose and highmaltose syrups. Such products are of considerable interest in theproduction of certain confectioneries because of the low hygroscoposity,low viscosity, good heat stability and mild, not too sweet taste ofmaltose. The industrial process of producing maltose syrups comprisesliquefying starch, then saccharification with a maltose producingenzyme, and optionally with an enzyme cleaving the 1.6- branching pointsin amylopectin, for instance an alpha.-1.6- amyloglucosidase.

The PS4 variants described here may be added to and thus become acomponent of a detergent composition. The detergent composition may forexample be formulated as a hand or machine laundry detergent compositionincluding a laundry additive composition suitable for pre-treatment ofstained fabrics and a rinse added fabric softener composition, or beformulated as a detergent composition for use in general household hardsurface cleaning operations, or be formulated for hand or machinedishwashing operations. In a specific aspect, we describe a detergentadditive comprising the PS4 variant. The detergent additive as well asthe detergent composition may comprise one or more other enzymes such asa protease, a lipase, a cutinase, an amylase, a carbohydrase, acellulase, a pectinase, a mannanase, an arabinase, a galactanase, axylanase, an oxidase, e.g., a laccase, and/or a peroxidase. In generalthe properties of the chosen enzyme(s) should be compatible with theselected detergent, (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

The PS4 variant may also be used in the production of lignocellulosicmaterials, such as pulp, paper and cardboard, from starch reinforcedwaste paper and cardboard, especially where repulping occurs at pH above7 and where amylases can facilitate the disintegration of the wastematerial through degradation of the reinforcing starch. The PS4 variantsmay especially be useful in a process for producing a papermaking pulpfrom starch-coated printed paper. The process may be performed asdescribed in WO 95/14807, comprising the following steps: a)disintegrating the paper to produce a pulp, b) treating with astarch-degrading enzyme before, during or after step a), and c)separating ink particles from the pulp after steps a) and b). The PS4variant may also be very useful in modifying starch where enzymaticallymodified starch is used in papermaking together with alkaline fillerssuch as calcium carbonate, kaolin and clays. With the PS4 variantsdescribed here it becomes possible to modify the starch in the presenceof the filler thus allowing for a simpler integrated process. A PS4variant may also be very useful in textile desizing. In the textileprocessing industry, amylases are traditionally used as auxiliaries inthe desizing process to facilitate the removal of starch-containing sizewhich has served as a protective coating on weft yams during weaving.Complete removal of the size coating after weaving is import-ant toensure optimum results in the subsequent processes, in which the fabricis scoured, bleached and dyed. Enzymatic starch break-down is preferredbecause it does not involve any harmful effect on the fiber material.The PS4 variant may be used alone or in combination with a cellulasewhen desizing cellulose-containing fabric or textile.

The PS4 variant may also be an amylase of choice for production ofsweeteners from starch A “traditional” process for conversion of starchto fructose syrups normally consists of three consecutive enzymaticprocesses, viz., a liquefaction process followed by a saccharificationprocess and an isomerization process. During the liquefaction process,starch is degraded to dextrins by an amylase at pH values between 5.5and 6.2 and at temperatures of 95-160° C. for a period of approx. 2hours. In order to ensure an optimal enzyme stability under theseconditions, 1 mM of calcium is added (40 ppm free calcium ions). Afterthe liquefaction process the dextrins are converted into dextrose byaddition of a glucoamylase and a debranching enzyme, such as anisoamylase or a pullulanase . Before this step the pH is reduced to avalue below 4.5, maintaining the high temperature (above 95° C.), andthe liquefying amylase activity is denatured. The temperature is loweredto 60° C., and glucoamylase and debranching enzyme are added. Thesaccharification process proceeds for 24-72 hours.

Feed Applications

In one embodiment, the PS4 variant polypeptide is capable of degradingresistant starch.

As used herein the term ‘degrading’ relates to the partial or completehydrolysis or degradation of resistant starch to glucose and/oroligosaccharides—such as maltose and/or—dextrins.

The PS4 variant polypeptide may degrade residual resistant starch thathas not been completely degraded by an animals amylase. By way ofexample, the PS4 variant polypeptide may be used to assist an animal'samylase (eg. pancreatic amylase) in improving the degradation ofresistant starch. Pancreatic α-amylase is excreted in the digestivesystem by animals. Pancreatic α-amylase degrades starch in the feed.However, a part of the starch, the resistant starch, is not degradedfully by the pancreatic a-amylase and is therefore not absorbed in thesmall intestine (see definition of resistant starch). The PS4 variantpolypeptide in some embodiments is able to assist the pancreatica-amylase in degrading starch in the digestive system and therebyincrease the utilisation of starch by the animal.

The ability of an enzyme to degrade resistant starch may be analysed forexample by a method developed and disclosed by Megazyme InternationalIreland Ltd. for the measurement of resistant starch, solubilised starchand total starch content of a sample (Resistant Starch Assay Procedure,AOAC Method 2002.02, AACC Method 32-40).

Accordingly, the PS4 variant polypeptides may be ingested by an animalfor beneficial purposes, and may therefore be incorporated into animalfeeds.

We therefore disclose the use of a PS4 variant polypeptide as acomponent for use in a feed comprising starch, or for use in a feedimprovement composition, in which the PS4 variant polypeptide is capableof degrading resistant starch. We also disclose a feed comprising astarch and a PS4 variant polypeptide. We further disclose a method ofdegrading resistant starch in a feed comprising contacting saidresistant starch with a PS4 variant polypeptide.

We further describe the use of a PS4 variant polypeptide in thepreparation of a feed comprising a starch, to degrade resistant starch.Furthermore, we disclose the use of a PS4 variant polypeptide in thepreparation of a feed to improve the calorific value of said feed. Wedisclose the use of an enzyme in the preparation of a feed to improveanimal performance. In a further embodiment, we describe a process forpreparing a feed comprising admixing a starch and a PS4 variantpolypeptide enzyme.

By way of example, use of a component comprising PS4 variantpolypeptides and which is capable of degrading resistant starch isadvantageous because there is a marked increase in the degradation ofstarch and/or starch degradation products in an animal. Furthermore,such use is advantageous because there is a marked increase in thedigestibility of starch and/or starch degradation products by an animal.Furthermore, such use is advantageous because it provides a means ofenhancing the efficiency of deriving energy from a feed by an animal.Furthermore, such use is advantageous because it provides a means toenhance the bioavailability of resistant starch.

Animal Feeds

Animal feeds for which the PS4 variant polypeptides are suitable for usemay be formulated to meet the specific needs of particular animal groupsand to provide the necessary carbohydrate, fat, protein and othernutrients in a form that can be metabolised by the animal.

Preferably, the animal feed is a feed for swine or poultry.

As used herein the term ‘swine’ relates to non-ruminant omnivores suchas pigs, hogs or boars. Typically, swine feed includes about 50 percentcarbohydrate, about 20 percent protein and about 5% fat. An example of ahigh energy swine feed is based on corn which is often combined withfeed supplements for example, protein, minerals, vitamins and aminoacids such as lysine and tryptophan. Examples of swine feeds includeanimal protein products, marine products, milk products, grain productsand plant protein products, all of which may further comprise naturalflavourings, artificial flavourings, micro and macro minerals, animalfats, vegetable fats, vitamins, preservatives or medications such asantibiotics.

It is to be understood that where reference is made in the presentspecification, including the accompanying claims, to ‘swine feed’ suchreference is meant to include “transition” or “starter” feeds (used towean young swine) and “finishing” or “grower” feeds (used following thetransition stage for growth of swine to an age and/or size suitable formarket).

As used herein the term ‘poultry’ relates to fowl such as chickens,broilers, hens, roosters, capons, turkeys, ducks, game fowl, pullets orchicks. Poultry feeds may be referred to as “complete” feeds becausethey contain all the protein, energy, vitamins, minerals, and othernutrients necessary for proper growth, egg production, and health of thebirds. However, poultry feeds may further comprise vitamins, minerals ormedications such as coccidiostats (for example Monensin sodium,Lasalocid, Amprolium, Salinomycin, and Sulfaquinoxaline) and/orantibiotics (for example Penicillin, Bacitracin, Chlortetracycline, andOxytetracycline).

Young chickens or broilers, turkeys and ducks kept for meat productionare fed differently from pullets saved for egg production. Broilers,ducks and turkeys have larger bodies and gain weight more rapidly thando the egg-producing types of chickens. Therefore, these birds are feddiets with higher protein and energy levels.

It is to be understood that where reference is made in the presentspecification, including the accompanying claims, to ‘poultry feed’ suchreference is meant to include “starter” feeds (post-hatching),“finisher”, “grower” or “developer” feeds (from 6-8 weeks of age untilslaughter size reached) and “layer” feeds (fed during egg production).

Animal feeds may be formulated to meet the animal's nutritional needswith respect to, for example, meat production, milk production, eggproduction, reproduction and response to stress. In addition, the animalfeeds are formulated to improve manure quality.

In a preferred aspect the animal feed contains a raw material such as alegume, for example pea or soy or a cereal, for example wheat, corn(maize), rye or barley. Suitably, the raw material may be potato. FEEDSTUFFS

The PS4 variant polypeptides may be used in feeds for animal consumptionby the indirect or direct application of the PS4 variant polypeptides tothe feed, whether alone or in combination with other ingredients, suchas food ingredients.

Typical food ingredients may include any one or more of an additive suchas an animal or vegetable fat, a natural or synthetic seasoning,antioxidant, viscosity modifier, essential oil, and/or flavour, dyeand/or colorant, vitamin, mineral, natural and/or non-natural aminoacid, nutrient, additional enzyme (including genetically manipulatedenzymes), a binding agent such as guar gum or xanthum gum, buffer,emulsifier, lubricant, adjuvant, suspending agent, preservative, coatingagent or solubilising agent and the like.

Examples of the application methods include, but are not limited to,coating the feed in a material comprising the PS4 variant polypeptide,direct application by mixing the PS4 variant polypeptide with the feed,spraying the PS4 variant polypeptide onto the feed surface or dippingthe feed into a preparation of the PS4 variant polypeptide.

The PS4 variant polypeptide is preferably applied by mixing it with afeed or by spraying onto feed particles for animal consumption.Alternatively, the PS4 variant polypeptide may be included in theemulsion of a feed, or the interior of solid products by injection ortumbling.

The PS4 variant polypeptide may be applied to intersperse, coat and/orimpregnate a feed. Mixtures with other ingredients may also be used andmay be applied separately, simultaneously or sequentially. Chelatingagents, binding agents, emulsifiers and other additives such as microand macro minerals, amino acids, vitamins, animal fats, vegetable fats,preservatives, flavourings, colourings, may be similarly applied to thefeed simultaneously (either in mixture or separately) or appliedsequentially.

Amount of PS4 Variant Polypeptide

The optimum amount of the PS4 variant polypeptide to be used will dependon the feed to be treated and/or the method of contacting the feed withthe PS4 variant polypeptide and/or the intended use for the same. Theamount of PS4 variant polypeptide should be in a sufficient amount to beeffective to substantially degrade resistant starch following ingestionand during digestion of the feed.

Advantageously, the PS4 variant polypeptide would remain effectivefollowing ingestion of a feed for animal consumption and duringdigestion of the feed until a more complete digestion of the feed isobtained, i.e. an increased calorific value of the feed is released.

Amylase Combinations

We disclose in particular combinations of PS4 variant polypeptides withamylases, in particular, maltogenic amylases. Maltogenic alpha-amylase(glucan 1,4-α-maltohydrolase, E.C. 3.2.1.133) is able to hydrolyzeamylose and amylopectin to maltose in the alpha-configuration, and isalso able to hydrolyze maltotriose as well as cyclodextrin.

A maltogenic alpha-amylase from Bacillus (EP 120 693) is commerciallyavailable under the trade name Novamyl (product of Novo Nordisk A/S,Denmark) and is widely used in the baking industry as an anti-stalingagent due to its ability to reduce retrogradation of starch. Novamyl isdescribed in detail in International Patent Publication WO 91/04669. Themaltogenic alpha-amylase Novamyl shares several characteristics withcyclodextrin glucanotransferases (CGTases), including sequence homology(Henrissat B, Bairoch A; Biochem. J., 316, 695-696 (1996)) and formationof transglycosylation products (Christophersen, C., et al., 1997,Starch, vol. 50, No. 1, 39-45).

In highly preferred embodiments, we disclose combinations comprising PS4variant polypeptides together with Novamyl or any of its variants. Suchcombinations are useful for baking, food production, or for otherpurposes. The Novamyl may in particular comprise Novamyl 1500 MG.

Other documents describing Novamyl and its uses include Christophersen,C., Pedersen, S., and Christensen, T., (1993) Method for production ofmaltose an a limit dextrin, the limit dextrin, and use of the limitdextrin. Denmark, and WO 95/10627. It is further described in U.S. Pat.No. 4,598,048 and U.S. Pat. No. 4,604,355. Each of these documents ishereby incorporated by reference, and any of the Novamyl polypeptidesdescribed therein may be used in combinations with any of the PS4variant polypeptides described here.

Variants, homologues, and mutants of Novamyl may be used for thecombinations, provided they retain alpha amylase activity. For example,any of the Novamyl variants disclosed in U.S. Pat. No. 6,162,628, theentire disclosure of which is hereby incorporated by reference, may beused in combination with the PS4 variant polypeptides described here. Inparticular, any of the polypeptides described in that document,specifically variants of SEQ ID NO: 1 of U.S. Pat. No. 6,162,628 at anyone or more positions corresponding to Q13, 116, D17, N26, N28, P29,A30, S32, Y33, G34, L35, K40, M45, P73, V74, D76 N77, D79, N86, R95,N99, 1N100, H103, Q1 19, N120, N131, S141, T142, A148, N152, A163, H169,N171, G172, 1174, N176, N187, F188, A192, Q201, N203, H220, N234, G236,Q247, K249, D261, N266, L268, R272, N275, N276, V279, N280, V281, D285,N287, F297, Q299, N305, K316, N320, L321, N327, A341, N342, A348, Q365,N371, N375, M378, G397, A381, F389, N401, A403, K425, N436, S442, N454,N468, N474, S479, A483, A486, V487, S493, T494, S495, A496, S497, A498,Q500, N507,1510, N513, K520, Q526, A555, A564, S573, N575, Q581, S583,F586, K589, N595, G618, N621, Q624, A629, F636, K645, N664 and/or T681may be used.

Amino Acid Sequences

The invention makes use of a PS4 variant nucleic acid, and the aminoacid sequences of such PS4 variant nucleic acids are encompassed by themethods and compositions described here.

As used herein, the term “amino acid sequence” is synonymous with theterm “polypeptide” and/or the term “protein”. In some instances, theterm “amino acid sequence” is synonymous with the term “peptide”. Insome instances, the term “amino acid sequence” is synonymous with theterm “enzyme”.

The amino acid sequence may be prepared/isolated from a suitable source,or it may be made synthetically or it may be prepared by use ofrecombinant DNA techniques.

The PS4 variant enzyme described here may be used in conjunction withother enzymes. Thus we further disclose a combination of enzymes whereinthe combination comprises a PS4 variant polypeptide enzyme describedhere and another enzyme, which itself may be another PS4 variantpolypeptide enzyme.

PS4 Nucleotide Sequence

As noted above, we disclose nucleotide sequences encoding the PS4variant enzymes having the specific properties described.

The term “nucleotide sequence” or “nucleic acid sequence” as used hereinrefers to an oligonucleotide sequence or polynucleotide sequence, andvariant, homologues, fragments and derivatives thereof (such as portionsthereof). The nucleotide sequence may be of genomic or synthetic orrecombinant origin, which may be double-stranded or single-strandedwhether representing the sense or anti-sense strand.

The term “nucleotide sequence” as used in this document includes genomicDNA, cDNA, synthetic DNA, and RNA. Preferably it means DNA, morepreferably cDNA sequence coding for a PS4 variant polypeptide.

Typically, the PS4 variant nucleotide sequence is prepared usingrecombinant DNA techniques (i.e. recombinant DNA). However, in analternative embodiment, the nucleotide sequence could be synthesised, inwhole or in part, using chemical methods well known in the art (seeCaruthers M H et al., (1980) Nuc Acids Res Symp Ser 215-23 and Horn T etal., (1980) Nuc Acids Res Symp Ser 225-232).

Preparation of Nucleic Acid Sequences

A nucleotide sequence encoding either an enzyme which has the specificproperties as defined herein (e.g., a PS4 variant polypeptide) or anenzyme which is suitable for modification, such as a parent enzyme, maybe identified and/or isolated and/or purified from any cell or organismproducing said enzyme. Various methods are well known within the art forthe identification and/or isolation and/or purification of nucleotidesequences. By way of example, PCR amplification techniques to preparemore of a sequence may be used once a suitable sequence has beenidentified and/or isolated and/or purified.

By way of further example, a genomic DNA and/or cDNA library may beconstructed using chromosomal DNA or messenger RNA from the organismproducing the enzyme. If the amino acid sequence of the enzyme or a partof the amino acid sequence of the enzyme is known, labelledoligonucleotide probes may be synthesised and used to identifyenzyme-encoding clones from the genomic library prepared from theorganism. Alternatively, a labelled oligonucleotide probe containingsequences homologous to another known enzyme gene could be used toidentify enzyme-encoding clones. In the latter case, hybridisation andwashing conditions of lower stringency are used.

Alternatively, enzyme-encoding clones could be identified by insertingfragments of genomic DNA into an expression vector, such as a plasmid,transforming enzyme-negative bacteria with the resulting genomic DNAlibrary, and then plating the transformed bacteria onto agar platescontaining a substrate for enzyme (i.e. maltose), thereby allowingclones expressing the enzyme to be identified.

In a yet further alternative, the nucleotide sequence encoding theenzyme may be prepared synthetically by established standard methods,e.g. the phosphoroamidite method described by Beucage S. L. et al.,(1981) Tetrahedron Letters 22, p 1859-1869, or the method described byMatthes et al., (1984) EMBO J. 3, p 801-805. In the phosphoroamiditemethod, oligonucleotides are synthesised, e.g. in an automatic DNAsynthesiser, purified, annealed, ligated and cloned in appropriatevectors.

The nucleotide sequence may be of mixed genomic and synthetic origin,mixed synthetic and cDNA origin, or mixed genomic and cDNA origin,prepared by ligating fragments of synthetic, genomic or cDNA origin (asappropriate) in accordance with standard techniques. Each ligatedfragment corresponds to various parts of the entire nucleotide sequence.The DNA sequence may also be prepared by polymerase chain reaction (PCR)using specific primers, for instance as described in U.S. Pat. No.4,683,202 or in Saiki R K et al., (Science (1988) 239, pp 487-491).

Variants/Homologues/Derivatives

We further describe the use of variants, homologues and derivatives ofany amino acid sequence of an enzyme or of any nucleotide sequenceencoding such an enzyme, such as a PS4 variant polypeptide or a PS4variant nucleic acid. Unless the context dictates otherwise, the term“PS4 variant nucleic acid” should be taken to include each of thenucleic acid entities described below, and the term “PS4 variantpolypeptide” should likewise be taken to include each of the polypeptideor amino acid entities described below.

Here, the term “homologue” means an entity having a certain homologywith the subject amino acid sequences and the subject nucleotidesequences. Here, the term “homology” can be equated with “identity”.

In the present context, a homologous sequence is taken to include anamino acid sequence which may be at least 75, 80, 85 or 90% identical,preferably at least 95, 96, 97, 98 or 99% identical to the subjectsequence. Typically, the homologues will comprise the same active sitesetc. as the subject amino acid sequence. Although homology can also beconsidered in terms of similarity (i.e. amino acid residues havingsimilar chemical properties/functions), in the context of this documentit is preferred to express homology in terms of sequence identity.

In the present context, an homologous sequence is taken to include anucleotide sequence which may be at least 75, 80, 85 or 90% identical,preferably at least 95, 96, 97, 98 or 99% identical to a nucleotidesequence encoding a PS4 variant polypeptide enzyme (such as a PS4variant nucleic acid). Typically, the homologues will comprise the samesequences that code for the active sites etc as the subject sequence.Although homology can also be considered in terms of similarity (i.e.amino acid residues having similar chemical properties/functions), inthe context of this document it is preferred to express homology interms of sequence identity.

Homology comparisons can be conducted by eye, or more usually, with theaid of readily available sequence comparison programs. Thesecommercially available computer programs can calculate % homologybetween two or more sequences. % homology may be calculated overcontiguous sequences, i.e. one sequence is aligned with the othersequence and each amino acid in one sequence is directly compared withthe corresponding amino acid in the other sequence, one residue at atime. This is called an “ungapped” alignment. Typically, such ungappedalignments are performed only over a relatively short number ofresidues.

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion will cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without penalising unduly the overall homology score. This isachieved by inserting “gaps” in the sequence alignment to try tomaximise local homology.

However, these more complex methods assign “gap penalties” to each gapthat occurs in the alignment so that, for the same number of identicalamino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimised alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons. For example when using the GCG Wisconsin Bestfitpackage the default gap penalty for amino acid sequences is −12 for agap and −4 for each extension.

Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (Devereux et al 1984 Nuc.Acids Research 12 p387). Examples of other software than can performsequence comparisons include, but are not limited to, the BLAST package(see Ausubel et al., 1999 Short Protocols in Molecular Biology, 4^(th)Ed—Chapter 18), FASTA (Altschul et al., 1990 J. Mol. Biol. 403-410) andthe GENEWORKS suite of comparison tools. Both BLAST and FASTA areavailable for offline and online searching (see Ausubel et al., 1999,Short Protocols in Molecular Biology, pages 7-58 to 7-60).

However, for some applications, it is preferred to use the GCG Bestfitprogram. A new tool, called BLAST 2 Sequences is also available forcomparing protein and nucleotide sequence (see FEMS Microbiol Lett 1999174(2): 247-50; FEMS Microbiol Lett 1999 177(1): 187-8 andtatiana@ncbi.nlm.nih.gov).

Although the final % homology can be measured in terms of identity, thealignment process itself is typically not based on an all-or-nothingpair comparison. Instead, a scaled similarity score matrix is generallyused that assigns scores to each pairwise comparison based on chemicalsimilarity or evolutionary distance. An example of such a matrixcommonly used is the BLOSUM62 matrix - the default matrix for the BLASTsuite of programs. GCG Wisconsin programs generally use either thepublic default values or a custom symbol comparison table if supplied(see user manual for further details). For some applications, it ispreferred to use the public default values for the GCG package, or inthe case of other software, the default matrix, such as BLOSUM62.

Alternatively, percentage homologies may be calculated using themultiple alignment feature in DNASIS (Hitachi Software), based on analgorithm, analogous to CLUSTAL (Higgins DG & Sharp PM (1988), Gene73(1), 237-244).

Once the software has produced an optimal alignment, it is possible tocalculate % homology, preferably % sequence identity. The softwaretypically does this as part of the sequence comparison and generates anumerical result.

The sequences may also have deletions, insertions or substitutions ofamino acid residues which produce a silent change and result in afunctionally equivalent substance. Deliberate amino acid substitutionsmay be made on the basis of similarity in amino acid properties (such aspolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues) and it is therefore useful to groupamino acids together in functional groups. Amino acids can be groupedtogether based on the properties of their side chain alone. However itis more useful to include mutation data as well. The sets of amino acidsthus derived are likely to be conserved for structural reasons. Thesesets can be described in the form of a Venn diagram (Livingstone C. D.and Barton G. J. (1993) “Protein sequence alignments: a strategy for thehierarchical analysis of residue conservation” Comput.Appl Biosci. 9:745-756)(Taylor W. R. (1986) “The classification of amino acidconservation” J. Theor.Biol. 119; 205-218). Conservative substitutionsmay be made, for example according to the table below which describes agenerally accepted Venn diagram grouping of amino acids. Set Sub-setHydrophobic FWYHKMILVAGC Aromatic FWYH Aliphatic ILV Polar WYHKREDCSTNQCharged HKRED Positively HKR charged Negatively ED charged SmallVCAGSPTND Tiny AGS

We further disclose sequences comprising homologous substitution(substitution and replacement are both used herein to mean theinterchange of an existing amino acid residue, with an alternativeresidue) that may occur i.e. like-for-like substitution such as basicfor basic, acidic for acidic, polar for polar etc. Non-homologoussubstitution may also occur i.e. from one class of residue to another oralternatively involving the inclusion of unnatural amino acids such asornithine (hereinafter referred to as Z), diaminobutyric acid ornithine(hereinafter referred to as B), norleucine ornithine (hereinafterreferred to as O), pyriylalanine, thienylalanine, naphthylalanine andphenylglycine.

Variant amino acid sequences may include suitable spacer groups that maybe inserted between any two amino acid residues of the sequenceincluding alkyl groups such as methyl, ethyl or propyl groups inaddition to amino acid spacers such as glycine or β-alanine residues. Afurther form of variation, involves the presence of one or more aminoacid residues in peptoid form, will be well understood by those skilledin the art. For the avoidance of doubt, “the peptoid form” is used torefer to variant amino acid residues wherein the α-carbon substituentgroup is on the residue's nitrogen atom rather than the α-carbon.Processes for preparing peptides in the peptoid form are known in theart, for example Simon R J et al., PNAS (1992) 89(20), 9367-9371 andHorwell D C, Trends Biotechnol. (1995) 13(4), 132-134.

The nucleotide sequences described here, and suitable for use in themethods and compositions described here (such as PS4 variant nucleicacids) may include within them synthetic or modified nucleotides. Anumber of different types of modification to oligonucleotides are knownin the art. These include methylphosphonate and phosphorothioatebackbones and/or the addition of acridine or polylysine chains at the 3′and/or 5′ ends of the molecule. For the purposes of this document, it isto be understood that the nucleotide sequences described herein may bemodified by any method available in the art. Such modifications may becarried out in order to enhance the in vivo activity or life span ofnucleotide sequences.

We further describe the use of nucleotide sequences that arecomplementary to the sequences presented herein, or any derivative,fragment or derivative thereof. If the sequence is complementary to afragment thereof then that sequence can be used as a probe to identifysimilar coding sequences in other organisms etc.

Polynucleotides which are not 100% homologous to the PS4 variantsequences may be obtained in a number of ways. Other variants of thesequences described herein may be obtained for example by probing DNAlibraries made from a range of individuals, for example individuals fromdifferent populations. In addition, other homologues may be obtained andsuch homologues and fragments thereof in general will be capable ofselectively hybridising to the sequences shown in the sequence listingherein. Such sequences may be obtained by probing cDNA libraries madefrom or genomic DNA libraries from other species, and probing suchlibraries with probes comprising all or part of any one of the sequencesin the attached sequence listings under conditions of medium to highstringency. Similar considerations apply to obtaining species homologuesand allelic variants of the polypeptide or nucleotide sequencesdescribed here.

Variants and strain/species homologues may also be obtained usingdegenerate PCR which will use primers designed to target sequenceswithin the variants and homologues encoding conserved amino acidsequences. Conserved sequences can be predicted, for example, byaligning the amino acid sequences from several variants/homologues.Sequence alignments can be performed using computer software known inthe art. For example the GCG Wisconsin PileUp program is widely used.

The primers used in degenerate PCR will contain one or more degeneratepositions and will be used at stringency conditions lower than thoseused for cloning sequences with single sequence primers against knownsequences.

Alternatively, such polynucleotides may be obtained by site directedmutagenesis of characterised sequences. This may be useful where forexample silent codon sequence changes are required to optimise codonpreferences for a particular host cell in which the polynucleotidesequences are being expressed. Other sequence changes may be desired inorder to introduce restriction enzyme recognition sites, or to alter theproperty or function of the polypeptides encoded by the polynucleotides.

The polynucleotides (nucleotide sequences) such as the PS4 variantnucleic acids described in this document may be used to produce aprimer, e.g. a PCR primer, a primer for an alternative amplificationreaction, a probe e.g. labelled with a revealing label by conventionalmeans using radioactive or non-radioactive labels, or thepolynucleotides may be cloned into vectors. Such primers, probes andother fiagments will be at least 15, preferably at least 20, for exampleat least 25, 30 or 40 nucleotides in length, and are also encompassed bythe term polynucleotides.

Polynucleotides such as DNA polynucleotides and probes may be producedrecombinantly, synthetically, or by any means available to those ofskill in the art. They may also be cloned by standard techniques.

In general, primers will be produced by synthetic means, involving astepwise manufacture of the desired nucleic acid sequence one nucleotideat a time. Techniques for accomplishing this using automated techniquesare readily available in the art.

Longer polynucleotides will generally be produced using recombinantmeans, for example using a PCR (polymerase chain reaction) cloningtechniques. The primers may be designed to contain suitable restrictionenzyme recognition sites so that the amplified DNA can be cloned into asuitable cloning vector.

Preferably, the variant sequences etc. are at least as biologicallyactive as the sequences presented herein.

As used herein “biologically active” refers to a sequence having asimilar structural function (but not necessarily to the same degree),and/or similar regulatory function (but not necessarily to the samedegree), and/or similar biochemical function (but not necessarily to thesame degree) of the naturally occurring sequence.

Hybridisation

We further describe sequences that are complementary to the nucleic acidsequences of PS4 variants or sequences that are capable of hybridisingeither to the PS4 variant sequences or to sequences that arecomplementary thereto.

The term “hybridisation” as used herein shall include “the process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing” as well as the process of amplification as carried out inpolymerase chain reaction (PCR) technologies. Therefore, we disclose theuse of nucleotide sequences that are capable of hybridising to thesequences that are complementary to the sequences presented herein, orany derivative, fragment or derivative thereof.

The term “variant” also encompasses sequences that are complementary tosequences that are capable of hybridising to the nucleotide sequencespresented herein.

Preferably, the term “variant” encompasses sequences that arecomplementary to sequences that are capable of hybridising understringent conditions (e.g. 50° C. and 0.2×SSC {1×SSC=0.15 M NaCl, 0.015M Na₃citrate pH 7.0}) to the nucleotide sequences presented herein.

More preferably, the term “variant” encompasses sequences that arecomplementary to sequences that are capable of hybridising under highstringent conditions (e.g. 65° C. and 0.1×SSC{1×SSC=0.15 M NaCl, 0.015 MNa₃citrate pH 7.0}) to the nucleotide sequences presented herein.

We further disclose nucleotide sequences that can hybridise to thenucleotide sequences of PS4 variants (including complementary sequencesof those presented herein), as well as nucleotide sequences that arecomplementary to sequences that can hybridise to the nucleotidesequences of PS4 variants (including complementary sequences of thosepresented herein). We further describe polynucleotide sequences that arecapable of hybridising to the nucleotide sequences presented hereinunder conditions of intermediate to maximal stringency.

In a preferred aspect, we disclose nucleotide sequences that canhybridise to the nucleotide sequence of a PS4 variant nucleic acid, orthe complement thereof, under stringent conditions (e.g. 50° C. and0.2×SSC). More preferably, the nucleotide sequences can hybridise to thenucleotide sequence of a PS4 variant, or the complement thereof, underhigh stringent conditions (e.g. 65° C. and 0.1×SSC).

Site-directed Mutagenesis

Once an enzyme-encoding nucleotide sequence has been isolated, or aputative enzyme-encoding nucleotide sequence has been identified, it maybe desirable to mutate the sequence in order to prepare an enzyme.Accordingly, a PS4 variant sequence may be prepared from a parentsequence.

Mutations may be introduced using synthetic oligonucleotides. Theseoligonucleotides contain nucleotide sequences flanking the desiredmutation sites.

A suitable method is disclosed in Morinaga et al., (Biotechnology (1984)2, p646-649). Another method of introducing mutations intoenzyme-encoding nucleotide sequences is described in Nelson and Long(Analytical Biochemistry (1989), 180, p 147-151). A further method isdescribed in Sarkar and Sommer (Biotechniques (1990), 8, p404-407—“Themegaprimer method of site directed mutagenesis”).

In one aspect the sequence for use in the methods and compositionsdescribed here is a recombinant sequence—i.e. a sequence that has beenprepared using recombinant DNA techniques.

These recombinant DNA techniques are within the capabilities of a personof ordinary skill in the art. Such techniques are explained in theliterature, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis,1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3,Cold Spring Harbor Laboratory Press.

In one aspect the sequence for use in the methods and compositionsdescribed here is a synthetic sequence—i.e. a sequence that has beenprepared by in vitro chemical or enzymatic synthesis. It includes, butis not limited to, sequences made with optimal codon usage for hostorganisms—such as the methylotrophic yeasts Pichia and Hansenula.

The nucleotide sequence for use in the methods and compositionsdescribed here may be incorporated into a recombinant replicable vector.The vector may be used to replicate and express the nucleotide sequence,in enzyme form, in and/or from a compatible host cell. Expression may becontrolled using control sequences eg. regulatory sequences. The enzymeproduced by a host recombinant cell by expression of the nucleotidesequence may be secreted or may be contained intracellularly dependingon the sequence and/or the vector used. The coding sequences may bedesigned with signal sequences which direct secretion of the substancecoding sequences through a particular prokaryotic or eukaryotic cellmembrane.

Expression of PS4 Nucleic Acids and Polypeptides

The PS4 polynucleotides and nucleic acids may include DNA and RNA ofboth synthetic and natural origin which DNA or RNA may contain modifiedor unmodified deoxy- or dideoxy- nucleotides or ribonucleotides oranalogs thereof. The PS4 nucleic acid may exist as single- ordouble-stranded DNA or RNA, an RNA/DNA heteroduplex or an RNA/DNAcopolymer, wherein the term “copolymer” refers to a single nucleic acidstrand that comprises both ribonucleotides and deoxyribonucleotides. ThePS4 nucleic acid may even be codon optimised to further increaseexpression.

The term “synthetic”, as used herein, is defined as that which isproduced by in vitro chemical or enzymatic synthesis. It includes but isnot limited to PS4 nucleic acids made with optimal codon usage for hostorganisms such as the the methylotrophic yeasts Pichia and Hansenula.

Polynucleotides, for example variant PS4 polynucleotides described here,can be incorporated into a recombinant replicable vector. The vector maybe used to replicate the nucleic acid in a compatible host cell. Thevector comprising the polynucleotide sequence may be transformed into asuitable host cell. Suitable hosts may include bacterial, yeast, insectand fungal cells.

The term “transformed cell” includes cells that have been transformed byuse of recombinant DNA techniques. The transformation typically occursby insertion of one or more nucleotide sequences into a cell that is tobe transformed. The inserted nucleotide sequence may be a heterologousnucleotide sequence (i.e. is a sequence that is not natural to the cellthat is to be transformed. In addition, or in the alternative, theinserted nucleotide sequence may be an homologous nucleotide sequence(i.e. is a sequence that is natural to the cell that is to betransformed)—so that the cell receives one or more extra copies of anucleotide sequence already present in it.

Thus in a further embodiment, we provide a method of making PS4 variantpolypeptides and polynucleotides by introducing a polynucleotide into areplicable vector, introducing the vector into a compatible host cell,and growing the host cell under conditions which bring about replicationof the vector. The vector may be recovered from the host cell.

Expression Constructs

The PS4 nucleic acid may be operatively linked to transcriptional andtranslational regulatory elements active in a host cell of interest. ThePS4 nucleic acid may also encode a fusion protein comprising signalsequences such as, for example, those derived from the glucoamylase genefrom Schwanniomyces occidentalis, α-factor mating type gene fromSaccharomyces cerevisiae and the TAKA-amylase from Aspergillus oryzae.Alternatively, the PS4 nucleic acid may encode a fusion proteincomprising a membrane binding domain.

Expression Vector

The PS4 nucleic acid may be expressed at the desired levels in a hostorganism using an expression vector.

An expression vector comprising a PS4 nucleic acid can be any vectorwhich is capable of expressing the gene encoding PS4 nucleic acid in theselected host organism, and the choice of vector will depend on the hostcell into which it is to be introduced. Thus, the vector can be anautonomously replicating vector, i.e. a vector that exists as anepisomal entity, the replication of which is independent of chromosomalreplication, such as, for example, a plasmid, a bacteriophage or anepisomal element, a minichromosome or an artificial chromosome.Alternatively, the vector may be one which, when introduced into a hostcell, is integrated into the host cell genome and replicated togetherwith the chromosome.

Components of the Expression Vector

The expression vector typically includes the components of a cloningvector, such as, for example, an element that permits autonomousreplication of the vector in the selected host organism and one or morephenotypically detectable markers for selection purposes. The expressionvector normally comprises control nucleotide sequences encoding apromoter, operator, ribosome binding site, translation initiation signaland optionally, a repressor gene or one or more activator genes.Additionally, the expression vector may comprise a sequence coding foran amino acid sequence capable of targeting the PS4 variant polypeptideto a host cell organelle such as a peroxisome or to a particular hostcell compartment. Such a targeting sequence includes but is not limitedto the sequence SKL. In the present context, the term ‘expressionsignal” includes any of the above control sequences, repressor oractivator sequences. For expression under the direction of controlsequences, the nucleic acid sequence the PS4 variant polypeptide isoperably linked to the control sequences in proper manner with respectto expression.

Preferably, a polynucleotide in a vector is operably linked to a controlsequence that is capable of providing for the expression of the codingsequence by the host cell, i.e. the vector is an expression vector. Theterm “operably linked” means that the components described are in arelationship permitting them to function in their intended manner. Aregulatory sequence “operably linked” to a coding sequence is ligated insuch a way that expression of the coding sequence is achieved undercondition compatible with the control sequences.

The control sequences may be modified, for example by the addition offurther transcriptional regulatory elements to make the level oftranscription directed by the control sequences more responsive totranscriptional modulators. The control sequences may in particularcomprise promoters.

Promotor

In the vector, the nucleic acid sequence encoding for the variant PS4polypeptide is operably combined with a suitable promoter sequence. Thepromoter can be any DNA sequence having transcription activity in thehost organism of choice and can be derived from genes that arehomologous or heterologous to the host organism.

Bacterial Promoters

Examples of suitable promoters for directing the transcription of themodified nucleotide sequence, such as PS4 nucleic acids, in a bacterialhost include the promoter of the lac operon of E. coli, the Streptomycescoelicolor agarase gene dagA promoters, the promoters of the Bacilluslicheniformis α-amylase gene (amyL), the promoters of the Bacillusstearothermophilus maltogenic amylase gene (amyM), the promoters of theBacillus amyloliquefaciens α-amylase gene (amyQ), the promoters of theBacillus subtilis xylA and xylB genes and a promoter derived from aLactococcus sp.-derived promoter including the P170 promoter. When thegene encoding the PS4 variant polypeptide is expressed in a bacterialspecies such as E. coli, a suitable promoter can be selected, forexample, from a bacteriophage promoter including a T7 promoter and aphage lambda promoter.

Fungal Promoters

For transcription in a fungal species, examples of useful promoters arethose derived from the genes encoding the, Aspergillus oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral α-amylase, A. niger acid stable α-amylase, A. nigerglucoamylase, Rhizomucor miehei lipase, Aspergillus oryzae alkalineprotease, Aspergillus oryzae triose phosphate isomerase or Aspergillusnidulans acetamidase.

Yeast Promoters

Examples of suitable promoters for the expression in a yeast speciesinclude but are not limited to the Gal 1 and Gal 10 promoters ofSaccharomyces cerevisiae and the Pichia pastoris AOX1 or AOX2 promoters.

Host Organisms

(I) Bacterial Host Organisms

Examples of suitable bacterial host organisms are gram positivebacterial species such as Bacillaceae including Bacillus subtilis,Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillusstearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens,Bacillus coagulans, Bacillus lautus, Bacillus megaterium and Bacillusthuringiensis, Streptomyces species such as Streptomyces murinus, lacticacid bacterial species including Lactococcus spp. such as Lactococcuslactis, Lactobacillus spp. including Lactobacillus reuteri, Leuconostocspp., Pediococcus spp. and Streptococcus spp. Alternatively, strains ofa gram-negative bacterial species belonging to Enterobacteriaceaeincluding E. coli, or to Pseudomonadaceae can be selected as the hostorganism.

(II) Yeast Host Organisms

A suitable yeast host organism can be selected from thebiotechnologically relevant yeasts species such as but not limited toyeast species such as Pichia sp., Hansenula sp or Kluyveromyces,Yarrowinia species or a species of Saccharomyces including Saccharomycescerevisiae or a species belonging to Schizosaccharomyce such as, forexample, S. Pombe species.

Preferably a strain of the methylotrophic yeast species Pichia pastorisis used as the host organism. Preferably the host organism is aHansenula species.

(III) Fungal Host Organisms

Suitable host organisms among filamentous fungi include species ofAspergillus, e.g. Aspergillus niger, Aspergillus oryzae, Aspergillustubigensis, Aspergillus awamori or Aspergillus nidulans. Alternatively,strains of a Fusarium species, e.g. Fusarium oxysporum or of aRhizomucor species such as Rhizomucor miehei can be used as the hostorganism. Other suitable strains include Thermomyces and Mucor species.

Protein Expression and Purification

Host cells comprising polynucleotides may be used to expresspolypeptides, such as variant PS4 polypeptides, fragments, homologues,variants or derivatives thereof. Host cells may be cultured undersuitable conditions which allow expression of the proteins. Expressionof the polypeptides may be constitutive such that they are continuallyproduced, or inducible, requiring a stimulus to initiate expression. Inthe case of inducible expression, protein production can be initiatedwhen required by, for example, addition of an inducer substance to theculture medium, for example dexamethasone or IPTG.

Polypeptides can be extracted from host cells by a variety of techniquesknown in the art, including enzymatic, chemical and/or osmotic lysis andphysical disruption. Polypeptides may also be produced recombinantly inan in vitro cell-free system, such as the TnT™ (Promega) rabbitreticulocyte system.

PS4 Antibodies

We also provide monoclonal or polyclonal antibodies to PS4 variantpolypeptides or fragments thereof. Thus, we further describe a processfor the production of monoclonal or polyclonal antibodies to an variantPS4 polypeptide, fragment, homologue, variant or derivative thereof.

If polyclonal antibodies are desired, a selected mammal (e.g., mouse,rabbit, goat, horse, etc.) is immunised with an immunogenic polypeptidebearing an epitope(s) from a polypeptide. Serum from the immunisedanimal is collected and treated according to known procedures. If serumcontaining polyclonal antibodies to an epitope from a polypeptidecontains antibodies to other antigens, the polyclonal antibodies can bepurified by immunoaffinity chromatography. Techniques for producing andprocessing polyclonal antisera are known in the art. In order that suchantibodies may be made, we also provide PS4 variant polypeptides orfragments thereof haptenised to another polypeptide for use asimmunogens in animals or humans.

Monoclonal antibodies directed against epitopes in the polypeptides canalso be readily produced by one skilled in the art. The generalmethodology for making monoclonal antibodies by hybridomas is wellknown. Immortal antibody-producing cell lines can be created by cellfusion, and also by other techniques such as direct transformation of Blymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus.Panels of monoclonal antibodies produced against epitopes in thepolypeptides can be screened for various properties; i.e., for isotypeand epitope affinity.

An alternative technique involves screening phage display librarieswhere, for example the phage express scFv fragments on the surface oftheir coat with a large variety of complementarity determining regions(CDRs). This technique is well known in the art.

Antibodies, both monoclonal and polyclonal, which are directed againstepitopes from polypeptides are particularly useful in diagnosis, andthose which are neutralising are useful in passive immunotherapy.Monoclonal antibodies, in particular, may be used to raise anti-idiotypeantibodies. Anti-idiotype antibodies are immunoglobulins which carry an“internal image” of the antigen of the agent against which protection isdesired.

Techniques for raising anti-idiotype antibodies are known in the art.These anti-idiotype antibodies may also be useful in therapy. For thepurposes of this document, the term “antibody”, unless specified to thecontrary, includes fiagments of whole antibodies which retain theirbinding activity for a target antigen. Such fragments include Fv, F(ab′)and F(ab′)₂ fragments, as well as single chain antibodies (scFv).Furthermore, the antibodies and fragments thereof may be humanisedantibodies, for example as described in EP-A-239400.

In preferred embodiments, the PS4 antibody is capable of specificallybinding to a particular PS4 variant polypeptide. Preferably, theantibody is capable of binding to a PS4 variant polypeptide underconsideration, but not to the wild type or parent polypeptide. Morepreferably, the antibody is capable of binding to the PS4 variantpolypeptide, but not to the parent or any other variant polypeptide. Inother words, preferred antibodies are those which are capable ofrecognising single amino acid changes in the sequence context of PS4.Such antibodies may be made by immunisation, as described above, andscreening for specific binding activity using dot blots, Western blots,etc, as known in the art.

REFERENCES

Penninga,D., van der Veen,B. A., Knegtel,R. M., van Hijum,S. A.,Rozeboom,H. J., Kalk,K. H., Dijkstra,B. W., Dijkhuizen,L. (1996). Theraw starch binding domain of cyclodextrin glycosyltransferase fromBacillus circulans strain 251. J.Biol.Chem. 271, 32777-32784.

Sambrook J,F.E.M.T. (1989). Molecular Cloning: A Laboratory Manual, 2ndEdn. Cold Spring Harbor Laboratory, Cold Spring Harbor N.Y.

Zhou,J. H., Baba,T., Takano,T., Kobayashi,S., Arai,Y. (1989). Nucleotidesequence of the maltotetraohydrolase gene from Pseudomonassaccharophila. FEBS Lett. 255, 37-41.

[1]. K. H. Park, Food Sci. Ind. 25 (1992) 73-82.

[2]. M. Okada and T. Nakakuki, Oligosaccharides: production, propertiesand application, in F. W. Schenck and R. E. Hebeda (Eds.), Starchhydrolysis products worldwide technology, production and application,VCH Publishers, New York, 1992, pp 335-366.

[3]. W. M.Fogarty, Microbial amylases, in W. M. Fogarty (Ed.), Microbialenzymes and biotechnology, Applied Science, London, 1983, pp. 1-92.

[4]. W. M. Fogarty and C. T. Kelly, Starch-degrading enzymes ofmicrobial origin, in M. J. Bull (Ed.), Progress in industrialmicrobiology, Vol. 15, Elsevier Scientific 1979, pp. 87-150.

[5]. K. Kainuma, S. Kobayashi, T. Ito, and S. Suzuki, FEBS Letters, 26(1972) 281-285.

[6]. N. Monma, T. Nakakuki, and K. Kainuma, Agric. Biol. Chem., 47(1983) 1769-1774.

[7]. J. F. Kennedy and C. A. White, Starch/Stärke 31 (1979) 93-99.

[8]. Y. Takasaki, Agric. Biol. Chem. 46 (1982) 1539-1547.

[9]. H. Taniguchi, C. M. Jae, N. Yoshigi, and Y. Maruyama, Agric. Biol.Chem. 47 (1983) 511-519.

[10]. H. Taniguchi, Maltohexaose-producing amylase of Bacillus circulansF-2 in R. B. Friedman (Ed.) Biotechnology of amylodextrinoligosaccharides. ACS Symp. Ser. 458. American Chemical Society,Washington DC, 1991, pp 111-124.

[11]. F. Bealin-Kelly, C. T. Kelly, and W. M. Fogarty, Biochem. Soc.Trans., 18 (1990) 310-311.

[12]. W. M. Fogarty, F. Bealin-Kelly, C. T. Kelly, and E. M. Doyle,Appl. Microbiol. Biotechnol., 36 (1991) 184-489.

[13]. N. Saito, Archives. Biochem. Biophys., 155 (1973) 290-298.

[14]. H. Okemoto, S. Kobayashi, M. Monma, H. Hashimoto, K. Hara, and K.Kainuma, Appl. Microbiol. Biotechnol., 25 (1986) 137-142.

[15]. O. Shida, T. Takano, H. Takagi, K. Kadowaki, and S. Kobayashi,Biosci., Biotechnol., Biochem. 56 (1992) 76-80.

[16]. (There is no ref. [16])

[17]. Y. Sakano, Y. Kashiwagi, and T. Kobayashi, Agric. Biol. Chem., 46(1982) 639-646.

[18]. Y. Takasaki, H. Shinohara, M. Tsuruhisa, S. Hayashi, K. Imada,Agric. Biol. Chem. 55 (1991) 1715-1720.

[19]. W. M. Fogarty, C. T. Kelly, A. C. Bourke, and E. M. Doyle,Biotechnol. Lett. 16 (1994) 473-478.

[20]. K. Wako, S. Hashimoto, S. Kubomura, A. Yokota, K. Aikawa, and J.Kamaeda, J. Jap. Soc. Starch. Sci 26 (1979) 175-181.

[21]. Y. Takasaki, Agric. Biol. Chem. 49 (1985) 1091-1097.

[22]. (There is no ref. [22])

[23]. T. Hayashi, T. Akiba, and K. Horikoshi, Appl. Microbiol.Biotechnol. 28 (1988b) 281-285.

[24]. G. Schmid, A. Candussio, and A. Bock, U.S. Pat. No. 5,304,723(1994).

[25] M. A. Mc Tigue, C. T. Kelly, E. M. Doyle, and W. M. Fogarty, EnzymeMicrob. Technol., 17 (1995) 570-573.

[26]. T. U. Kim, B. G. Gu, J. Y. Jeong, S. M. Byun, and Y. C. Shin,Appl. Environm. Microbiol. 61 (1995) 3105-3112.

EXAMPLES Example 1 Cloning of PS4

Pseudomonas sacharophila is grown overnight on LB media and chromosomalDNA is isolated by standard methods (Sambrook J, 1989). A 2190 bpfragment containing the PS4 openreading frame (Zhou et al., 1989) isamplified from P. sacharophila chromosomal DNA by PCR using the primersP1 and P2 (see Table 1). The resulting fragment is used as a template ina nested PCR with primers P3 and P4, amplifying the openreading frame ofPS4 without its signal sequence and introducing a NcoI site at the 5′end of the gene and a BamHI site at the 3′end. Together with the NcoIsite a codon for a N-terminal Methionine is introduced, allowing forintracellular expression of PS4. The 1605 bp fragment is cloned intopCRBLUNT TOPO (Invitrogen) and the integrity of the construct analysedby sequencing.

The E. coli Bacillus shuttle vector pDP66K (Penninga et al., 1996) ismodified to allow for expression of the PS4 under control of the P32promoter and the ctgase signal sequence. The resulting plasmid, pCSmta(see FIG. 1) is transformed into B. subtilis.

A second expression construct is made in which the starch binding domainof PS4 is removed. In a PCR with primers P3 and P6 (Table 1) on pCSmta,a truncated version of the mta gene is generated. The full length mtagene in pCSmta is exchanged with the truncated version which resulted inthe plasmid pCSmta-SBD (FIG. 1)

Example 2 Site Directed Mutagenesis

Mutations are introduced into the mta gene by 2 methods. Either by a 2step PCR based method, or by a Quick Exchange method (QE). Forconvenience the mta gene is split up in 3 parts; a PvuI-FspI fragment, aFspI-PstI fragment and a PstI-AspI fragment, further on referred to asfragment 1, 2 and 3 respectively.

In the 2 step PCR based method, mutations are introduced using Pfu DNApolymerase (Stratagene). A first PCR is carried out with a mutagenesisprimer (Table 2) for the coding strand plus a primer downstream on thelower strand (either 2R or 3R Table 1). The reaction product is used asa primer in a second PCR together with a primer upstream on the codingstrand. The product of the last reaction is cloned into pCRBLUNT topo(Invitrogen) and after sequencing the fragment is exchanged with thecorresponding fragment in pCSmta.

Using the Quick Exchange method (Stratagene), mutations are introducedusing two complementary primers in a PCR on a plasmid containing the mtagene, or part of the mta gene.

For this purpose a convenient set of plasmids is constructed, comprisingof 3 SDM plasmids and 3 pCSA plasmids (Figure2). The SDM plasmids eachbear I of the fragments of the mta gene as mentioned above, in which thedesired mutation is introduced by QE. After verification by sequencing,the fragments are cloned into the corresponding recipient pCSA plasmid.The pCSA plamids are inactive derivatives from pCSmta. Activity isrestored by cloning the corresponding fragment from the SDM plasmid,enabling easy screening. TABLE 1 Primers used in cloning the mta gene,and standard primers used in construction of site directed mutants withthe 2 step PCR method. Introduced Primer Primer sequence site P1 5′- ATGACG AGG TCC TTG TTT TTC (SEQ ID NO: 22) P2 5′- CGC TAG TCG TCC ATG TCG(SEQ ID NO: 23) P3 5′- GCC ATG GAT CAG GCC GGC AAG AGC CCG (SEQ ID NO:24) NcoI P4 5′- TGG ATC CTC AGA ACG AGC CGC TGG T (SEQ ID NO: 25) BamHIP6 5′- GAA TTC AGC CGC CGT CAT TCC CGC C (SEQ ID NO: 26) EcoRI 2L 5′-AGA TTT ACG GCA TGT TTC GC (SEQ ID NO: 27) 2R 5′- TAG CCG CTA TGG AAGCTG AT (SEQ ID NO: 28) 3L 5′- TGA CCT TCG TCG ACA ACC AC (SEQ ID NO: 29)3R 5′- GAT AGC TGC TGG TGA CGG TC (SEQ ID NO: 30)

TABLE 2 Primers used to introduce site directed mutations in mta. Ifused in the Quick Exchange method, a complementary lower-strand primeris used in combination with the mentioned primer. The template on whichthe PGR is run is indicated and the used method. Mutations are indicatedin bold, and introduced or removed restriction sites underlined andmentioned. Introduced Mutant Template Method Primer (upper) site G69PSDM1 QE AGC TGG ACC GAC CCG GGC AAG TCC +XmaI GGC (SEQ ID NO: 31) G103PSDM1 QE GCC GGC GCA CTC CCT GGC GCC GGG −DraIII GTG (SEQ ID NO: 32)G121P SDM1 QE CAC ATG AAC CGC CC G TAC CCG GAC −SacII AAG (SEQ ID NO:33) G132P SDM1 QE CAA CCT GCC GGC CCC GCA GGG CTT −FseI CTG G (SEQ IDNO: 34) A141P SDM1 QE CGC AAC GAC TGC CCG GAT CCG GGC +BamHI AAC (SEQ IDNO: 35) S161P SDM1 QE ATC GGC GGC GAG CCA GAT CTG AAC +BglII ACC (SEQ IDNO: 36) A199P SDM2 QE GTT CGC GGC TAT CCG CCC GAG CGG — GTC (SEQ ID NO:37) S213P SDM2 QE GAC AGC GCC GAC CCA AGC TTC TGC GTT (SEQ ID NO: 38)G223A SDM2 QE GAG CTG TGG AAA GCC CCT TCT GAA — TAT C (SEQ ID NO: 39)A268P pGSmta 2PGR AAC GGC TCG GTC CCG GAC TGG AAG — CAT (SEQ ID NO: 40)G313P pGSmta 2PGR GCG CTG CAG GAC CC G CTG ATC CGC +EcoO109 CAG (SEQ IDNO: 41) G342P pGSmta 2PGR GAC TGG GGC TAC CCG GAC TTC ATC — CGC (SEQ IDNO: 42) S367P SDM3 QE GCG AT A AGC TTC CAT CCG GGC TAC +HinDIII AGC (SEQID NO: 43) S399P SDM3 QE GGC CAG GTT GCC CCG GG A AGC TT C +HinDIII AGC(SEQ ID NO: 44) G400P SDM3 QE CAG GTT GCC AGC CCG AGC TTC AGC +AvaI GAG(SEQ ID NO: 45)

TABLE 3 Constructed mutants. In most of the site directed mutants arestriction site is added, allowing for quick identification. Sitedirected mutants are either created by a Quick Exchange method (QE) orby a 2 PCR based method as described above. Mutant Identification 2PCR/QE cloned in; Location G69P +XmaI QE pCSΔ1 1 G103P −DraIII QEpCS-mta 1 G121P −SacII QE pCS-mta 1 G132P −FseI QE pCS-mta 1 A141P+BamHI QE pCS-mta 1 S161P +BglII QE pCSΔ1 1 A199P — QE pCSAΔ 2 S213P+HinDIII QE pCSΔ2 2 G223A — QE pCSΔ2 2 A268P — 2PCR pCSΔ2 2 G313P+EcoO109 2PCR pCSΔ3 3 G342P — 2PCR pCSΔ3 3 S367P +HinDIII QE pCSΔ3 3S399P +HinDIII QE pCSΔ3 3 G400P +AvaI QE pCSΔ3 3

TABLE 4 Features of the SDM and pCSA plasmids PlasmidFeatures/construction SDM1 pBlueSK + 480 bp SalI-StuI fragment mta SDM2pBlueSK + 572 bp SacII-PstI fragment mta SDM3 pBlueSK + 471 bp Sali-Stuifragment mta pCSΔ1 FseI site filled in with Klenow ----> frameshift inmta pCSΔ2 FspI-PstI fragment of mta replaced with ‘junk-DNA’ pCSΔ3PstI-AspI fragment of mta replaced with ‘junk-DNA’

Bacillus subtilis (strain DB104A; Smith et al. 1988; Gene 70, 351-361)is transformed with the mutated pCS-plasmids according to the followingprotocol.

A. Media for Protoplasting and Transformation 2 × SMM per litre: 342 gsucrose (1 M); 4.72 g sodium maleate (0.04 M); 8:12 g MgC1₂, 6H₂0 (0.04M); pH 6.5 with concentrated NAOH. Distribute in 50-ml portions andautoclave for 10 min 4 ×YT 2 g Yeast extract + 3.2 g Tryptone + 0.5 gNaCl per 100 ml. (½ NaCl) mix equal volumes of 2 × SMM and 4 × YT. SMMP10 g polyethyleneglycol 6000 (BDH) or 8000 (Sigma) in PEG 25 ml 1 × SMM(autoclave for 10 min.).

B. Media for Plating/regeneration agar 4% Difco minimal agar. Autoclavefor 15 min. sodium succinate 270 g/l (1 M), pH 7.3 with HCl. Autoclavefor 15 min. phosphate buffer 3.5 g K₂HPO₄ + 1.5 g KH₂PO₄ per 100 ml.Autoclave for 15 min. MgCl₂ 20.3 g MgCl₂, 6H₂0 per 100 ml (1 M).casamino acids 5% (w/v) solution. Autoclave for 15 min. yeast extract 10g per 100 ml, autoclave for 15 min. glucose 20% (w/v) solution.Autoclave for 10 min. DM3 regeneration mix at 60 C. (waterbath; 500-mlbottle): medium: 250 ml sodium succinate 50 ml casamino acids 25 mlyeast extract 50 ml phosphate buffer 15 ml glucose 10 ml MgCl₂ 100 mlmolten agar Add appropriate antibiotics: chloramphemcol andtetracycline, 5 ug/ml; erythromycin, 1 ug/ml. Selection on kanamycin isproblematic in DM3 medium: concentrations of 250 ug/ml may be required.

C. Preparation of Protoplasts

-   -   1. Use detergent-free plastic or glassware throughout.    -   2. Inoculate 10 ml of 2×YT medium in a 100-ml flask from a        single colony. Grow an overnight culture at 25-30 C in a shaker        (200 rev/min).    -   3. Dilute the overnight culture 20 fold into 100 ml of fresh        2×YT medium (250-ml flask) and grow until OD₆₀₀=0.4-0.5 (approx.        2h) at 37C in a shaker (200-250 rev/min).    -   4. Harvest the cells by centrifugation (9000 g, 20 min, 4 C).    -   5. Remove the supernatant with pipette and resuspend the cells        in 5 ml of SMMP +5 mg lysozyme, sterile filtered.    -   6. Incubate at 37 C in a waterbath shaker (100 rev/min).

After 30 min and thereafter at 15 min intervals, examine 25 ul samplesby microscopy. Continue incubation until 99% of the cells areprotoplasted (globular appearance).

Harvest the protoplasts by centrifugation (4000g, 20 min, RT) and pipetoff the supernatant. Resuspend the pellet gently in 1-2 ml of SMMP.

The protoplasts are now ready for use. (Portions (e.g. 0.15 ml) can befrozen at −80 C for future use (glycerol addition is not required).Although this may result in some reduction of transformability, 106transformants per ug of DNA can be obtained with frozen protoplasts).

D. Transformation

-   -   1. Transfer 450 ul of PEG to a microtube.    -   2. Mix 1-10 ul of DNA (0.2 ug) with 150 ul of protoplasts and        add the mixture to the microtube with PEG. Mix immediately, but        gently.    -   3. Leave for 2 min at RT, and then add 1.5 ml of SMMP and mix.    -   4. Harvest protoplasts by microfuging (10 min, 13.000 rev/min        (10-12.000 g)) and pour off the supernatant. Remove the        remaining droplets with a tissue.

Add 300 ul of SMMP (do not vortex) and incubate for 60-90 min at 37 C ina waterbath shaker (100 rev/min) to allow for expression of antibioticresistance markers. (The protoplasts become sufficiently resuspendedthrough the shaking action of the waterbath.)

Make appropriate dilutions in 1×SSM and plate 0.1 ml on DM3 plates

Example 4 Fermentation of PS4 Variants in Shake Flasks

The shake flask substrate is prepared as follows: Ingredient % (w/v)Water — Yeast extract 2 Soy Flour 2 NaCl 0.5 Dipotassium phosphate 0.5Antifoam agent 0.05

The substrate is adjusted to pH 6.8 with 4N sulfuric acid or sodiumhydroxide before autoclaving. 100 ml of substrate is placed in a 500 mlflask with one baffle and autoclaved for 30 minutes. Subsequently, 6 mlof sterile dextrose syrup is added.

The dextrose syrup is prepared by mixing one volume of 50% w/v dextrosewith one volume of water followed by autoclaving for 20 minutes.

The shake flask are inoculated with the variants and incubated for 24hours at 35° C./ 180rpm in an incubator. After incubation cells areseparate from broth by centrifugation (10.000× g in 10 minutes) andfinally, the supernatant is made cell free by microfiltration at 0,2 μm.

The cell free supernatant is used for assays and application tests.

Example 5 Exo-amylase Assay

One unit is defined as activity degrading 1 umol per 1 min. ofPNP-coupled maltopentaose so that 1 umol PNP per 1 min. can be releasedby excess a-glucosidase in the assay mix.

The assay mix contains 50 ul 50 mM Na-citrate, 5 mM CaCl2, pH 6,5 with25 ul enzyme sample and 25 ul Betamyl substrate (Glc5-PNP anda-glucosidase) from Megazyme, Ireland (1 vial dissolved in 10 ml water).

The assay mix is incubated for 30 min. at 40C and then stopped by adding150 ul 4% Tris.

Absorbance at 420 nm is measured using an ELISA-reader and the activityis calculate based on Activity=A420 *d * 0,0351 U/ml enzyme sample.

Example 6 Half-life Determination

Definition

t½ is the time (in minutes) during which half the enzyme activity isinactivated under defined heat conditions.

Principle: In order to determine the half life of the enzyme, the sampleis heated for 1-10 minutes at 60° C. or higher. The half life value willthen be calculated by measuring the residual amylase activity.

Reagents: Buffer: 50 mM Citric acid, 5 mM CaCl₂, pH 6.5. Dissolve 10.5 gcitric acid in dH₂0, add 5 ml IM CaCI₂ and adjust pH to 6.5 with 2MNaOH. Substrate: Exo-amylase reaction mix according to assay procedure.

Equipment: Eppendorf Heat Incubator (Termomixer comfort), Mutipipette,ELISA reader.

Temperature: 60° C or. higher up to 90° C

Procedure: In an Eppendorf vial, 1000 μl buffer is preheated for atleast 10 minutes at 60° C. or higher. The heat treatment of the sampleis started addition of 100 μl of the sample to the preheated bufferunder continuous mixing (800 rpm). After 0, 2, 4, 6, 8 and 9 minutes ofincubation, the treatment is stopped by transferring 45 l of the sampleto 1000 l of the buffer equilibrated at 20° C. and incubating for oneminute at 1500 rpm and at 20° C.. The residual activity is measured withthe exo-amylase assay.

Calculation: Calculation of 1½ is based on the slope of loglO (thebase-10 logarithm) of the residual amylase activity versus theincubation time. tl/₂ is calculated as slope/0.301=tt2½

Example 7 Tests

Preparation of Doughs

The doughs are made in the Farinograph at 30.0° C. 10.00 g reformedflour is weighed out and added in the Farinograph; after 1 min. thereference/sample (reference=buffer or water, sample=enzyme+buffer orwater) is added with a sterile pipette through the holes of the kneadingvat. After 30 sec. the flour is scraped off the edges—also through theholes of the kneading vat. The sample is kneaded for 7 min.

A test with buffer or water is performed on the Farinograph before thefinal reference is run. FU should be 400 on the reference, if it is not,this should be adjusted with, for example, the quantity of liquid.

The reference/sample is removed with a spatula and placed in the hand(with a disposable glove on it), before it is filled into small glasstubes (of approx. 4.5 cm's length) that are put in NMR tubes and corkedup. 7 tubes per dough are made.

The Farinograph is cleaned with demineralised water and wiped withKimwipes between each test.

When all the samples have been run, the tubes are placed in a(programmable) water bath at 33° C. (without corks) for 25 min. andhereafter the water bath is set to profile 4 or 5.

Profile 4:5 min. at 33° C., then the water bath is heated to 98° C over.56 min. (1.1° C. per minute) and kept at 96° C. for 5 min. This is tomake sure that the 95° C. are reached. Method 1- M1.

After 7 days of storage at 20.0° C. in a thermo cupboard 10-20 mgsamples of crumb weighed out and placed in 40 μl aluminium standard DSCcapsules. 10 capsules are prepared per treatment and kept at 20° C.

The capsules are used for Differential Scanning Calorimetry on a MetlerToledo DSC (XX). As parameters are used a heating cycle of 20-95° C.with 10° C. per minute. Gas/flow: N₂/80 ml per minute.

When all the capsules have been tested, the results are peaked and thefollowing parameters are calculated: Integral: mJ; Normalized: Jg{acuteover ( )}−1, Onset: ° C.

Example 8 Thermostability Results

When analysed for thermostability the following half-lifes aredetermined for the variants and wild type PS4 as shown in Table 5. TABLE5 Half-lifes at 60° C. determined for wild type PS4 and variantscontaining the listed mutations Mutation t½ (60° C.) 1 Wild type PS4 2.12 G69P 2.5 3 G103P 2.3 4 G121P 1.8 5 G132P nd 6 A141P 8.0 7 S161P nd 8A199P 1.3 9 S213P 2.2 10 G223A 3.2 11 A268P 3.4 12 G313P 2.5 13 G342P nd14 S367P 1.9 15 S399P 4.4 16 G400P 2.5

Example 9 Antistaling Effects

Model bread crumbs are prepared and measured according to Example 7. Asshown in FIG. 3, PS4 variants with mutations A141P and G223A show areduction of the amylopectin retrogradation after baking as measured byDifferential Scanning Calorimetry in comparison to wild type PS4(PS4ccl). The variant A141P with increased thermostability also shows anincreased dosage effect in contrast to wild type PS4 (PS4cc1).

Example 10 Control of volume of Danish Rolls

Danish Rolls are prepared from a dough based on 2000 g Danish reformflour (from Cerealia), 120 g compressed yeast, 32 g salt, and 32 gsucrose. Water is added to the dough according to prior wateroptimisation.

The dough is mixed on a Diosna mixer (2 min. at low speed and 5 min. athigh speed). The dough temperature after mixing is kept at 26° C. 1350 gdough is scaled and rested for 10 min. in a heating cabinet at 30° C.The rolls are moulded on a Fortuna molder and proofed for 45 min. at 34°C. and at 85% relative humidity. Subsequently the rolls are baked in aBago 2 oven for 18 min. at 250° C. with steam in the first 13 seconds.After baking the rolls are cooled for 25 min. before weighing andmeasuring of volume.

The rolls are evaluated regarding crust appearance, crumb homogeneity,caping of the crust, ausbund and specific volume (measuring the volumewith the rape seed displacement method).

Based on these criteria it is found that the PS4 variant A141P (dosed at0.1-0.2 mg per kg of flour) increases the specific volume and improvethe quality parameters of Danish rolls. Thus PS4 variants are able tocontrol the volume of baked products.

REFERENCES

Each of the applications and patents mentioned in this document, andeach document cited or referenced in each of the above applications andpatents, including during the prosecution of each of the applicationsand patents (“application cited documents”) and any manufacturer'sinstructions or catalogues for any products cited or mentioned in eachof the applications and patents and in any of the application citeddocuments, are hereby incorporated herein by reference. Furthermore, alldocuments cited in this text, and all documents cited or referenced indocuments cited in this text, and any manufacturer's instructions orcatalogues for any products cited or mentioned in this text, are herebyincorporated herein by reference.

Various modifications and variations of the described methods and systemof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the claims.

1. A PS4 variant polypeptide derivable from a parent polypeptide, theparent polypeptide having non-maltogenic exoamylase activity, which PS4variant polypeptide comprises an alanine substitution at position 223with reference to the position numbering of a Pseudomonas saccharophiliaexoamylase sequence shown as SEQ ID NO: 1, in which the PS4 variantpolypeptide has at least 95% homology to SEQ ID NO: 1 and non-maltogenicexoamylase activity and a higher thermostability compared to the parentpolypeptide when tested under the same conditions.
 2. A PS4 variantpolypeptide according to claim 1, in which the parent polypeptidecomprises a glucan 1,4-alpha-maltotetrahydrolase (EC 3.2.1.60).
 3. A PS4variant polypeptide according to claim 2, in which the parentpolypeptide is a non-maltogenic exoamylase of Pseudomonas saccharophiliaor Pseudomonas stutzeri.
 4. A PS4 variant polypeptide according to claim3, in which the parent polypeptide is a non-maltogenic exoamylase fromPseudomonas saccharophilia having a sequence shown as SEQ ID NO: 1 orSEQ ID NO:
 9. 5. A PS4 variant polypeptide according to claim 1, inwhich the thermostability is measured by establishing a half life(t_(1/2)), at 60 degrees C.
 6. A PS4 variant polypeptide according toclaim 1, which has a higher pH stability compared to the parentpolypeptide when tested under the same conditions, wherein the pH isbetween pH5 to pH10.5.
 7. A PS4 variant polypeptide according to claim6, which has 10% or more pH stability compared to the parentpolypeptide, wherein the pH is between pH5 to pH10.5.
 8. A PS4 variantpolypeptide according to claim 1, which comprises a sequence PSac-G223A(SEQ ID NO: 4).
 9. A PS4 variant polypeptide according to claim 5, inwhich the half life (t_(1/2)) at 60 degrees C. is increased by 15% ormore relative to the parent polypeptide.
 10. A PS4 variant polypeptideaccording to claim 5, in which the half life (ti_(1/2)) at 60 degrees C.is increased by 50% or more relative to the parent polypeptide.
 11. APS4 variant polypeptide according to claim 5, in which the half life(t_(1/2)) at 60 degrees C is increased by 100% or more relative to theparent polypeptide.
 12. A PS4 variant polypeptide according to claim 1,which has a higher activity compared to the parent polypeptide.
 13. APS4 variant polypeptide according to claim 12, in which the activity istested using a waxy maize starch incubation test.
 14. A PS4 variantpolypeptide according to claim 6, which has 20% or more pH stabilitycompared to the parent polypeptide.
 15. A PS4 variant polypeptideaccording to claim 6, which has 50% or more pH stability compared to theparent polypeptide.
 16. A PS4 variant polypeptide according to claim 1,in which the polypeptide is obtainable by altering the sequence of aparent polypeptide having non-maltogenic exoamylase activity, byintroducing an alanine substitution at position 223 with reference tothe position numbering of a Pseudomonas saccharophilia exoamylasesequence shown as SEQ ID NO: 1 such that the polypeptide has a higherthermostability compared to the parent polypeptide when tested under thesame conditions.
 17. A polypeptide according to claim 16, which isobtainable by altering the sequence of a nucleic acid which encodes theparent polypeptide.
 18. A combination of a PS4 variant polypeptideaccording to claim 1, together with Novamyl, or a variant, homologue, ormutants thereof which has alpha amylase activity, or a compositioncomprising such.
 19. A PS4 variant polypeptide derivable from a parentpolypeptide, the parent polypeptide being a non-maltogenic exoamylasefrom Pseudomonas saccharophilia, the variant having a sequence shown asSEQ ID NO: I and non-maltogenic exoamylase activity and comprising analanine substitution at position 223, or a polypeptide which has atleast 95% similarity thereto, non-maltogenic exoamylase activity and analanine substitution at position
 223. 20. A combination of a PS4 variantpolypeptide according to claim 19, together with Novamyl, or a variant,homologue, or mutants thereof which has alpha amylase activity, or acomposition comprising such.
 21. A polypeptide obtainable by alteringthe sequence of a non-maltogenic exoamylase from Pseudomonassaccharophilia having a sequence shown as SEQ ID NO: 1, or a polypeptidewhich has at least 95% similarity thereto, by introducing an alaninesubstitution at position
 223. 22. A polypeptide according to claim 19which is obtainable by altering the sequence of a nucleic acid whichencodes the non-maltogenic exoamylase from Pseudomonas saccharophilia.23. A combination of a PS4 variant polypeptide according to claim 21,together with Novamyl, or a variant, homologue, or mutants thereof whichhas alpha amylase activity, or a composition comprising such.